Method for the synthesis of pyrrole and imidazole carboxamides on a solid support

ABSTRACT

The present invention describes a novel method for the solid phase synthesis of polyamides containing imidazole and pyrrole carboxamides. The polyamides are prepared on a solid support from aromatic carboxylic acids and aromatic amines with high stepwise coupling yields (&gt;99%), providing milligram quantities of highly pure polyamides. The present invention also describes the synthesis of analogs of the natural products Netropsin and Distamycin A, two antiviral antibiotics. The present invention also describes a novel method for the solid phase synthesis of imidazole and pyrrole carboxamide polyamide-oligonucleotide conjugates. This methodology will greatly increase both the complexity and quantity of minor-groove binding polyamides and minor-groove binding polyamide-oligonucleotide conjugates which can be synthesized and tested.

This work was partially supported by the United States Governmentthrough the National Institute of Health under Grant No. GM 27681. TheUnited States Government may have certain rights in this invention.

FIELD OF THE INVENTION

This invention relates to the field of peptide chemistry. Specifically,this invention relates to a novel process for preparing polyamides andpolyamide conjugates containing imidazole and pyrrole carboxamides usingsolid state chemistry. Also included in this invention is a simple andeffective method for preparing analogs of the antiviral antibioticsNetropsin and Distamycin A.

BACKGROUND OF THE INVENTION

Proteins and peptides play a critical role in virtually all biologicalprocesses, functioning as enzymes, hormones, antibodies, growth factors,ion carriers, antibiotics, toxins, and neuropeptides. Biologicallyactive proteins and peptides, therefore, have been a major target forchemical synthesis. Chemical synthesis is used to verify structure andto study the relationship between structure and function, with the goalof designing novel compounds for potential therapeutic use. Thus,modified or novel peptides may be synthesized which have improvedtherapeutic activity and/or reduced side effects.

There are two basic methods for synthesizing proteins and peptides: thechemistry is either carried out in solution (solution phase) or on asolid support (solid phase). A major disadvantage of solution phasesynthesis of peptides is the poor solubility of the protected peptideintermediates in organic solvents. Additionally, solution phasesynthesis requires extensive experience on the part of the scientist andthe purifications are difficult and time consuming. Solid phasesynthesis overcomes these problems and thus, has become the method ofchoice in synthesizing peptides and proteins.

The basic approach for solid phase peptide synthesis is illustrated inFIG. 1. Briefly, the carboxy-terminal amino acid of the peptide to besynthesized is protected and covalently attached to a solid support,typically a resin. The subsequent amino acids (which have also beenprotected) are then sequentially added. When the synthesis is completethe peptide is deprotected, cleaved from the resin and purified. Becausethe molecules being synthesized are so large it is imperative that thesteps proceed rapidly, in high yields and with minimal side reactions.

The most commonly used solid supports are cross-linked polystyrene andpolydimethylacrylamide resins, which are both derivatives ofpolyethylene. In 1978, Merrifield and coworkers introduced thetert-butyloxycarbonylaminoacyl-4-(oxymethyl)phenyl-acetamidomethyl-resin(PAM resin), a novel polystyrene resin for solid phase peptide synthesis(Mitchell et al. (1978) J. Org. Chem. 43:2845-2852). PAM resin has apreformed resin ester linkage which is stable to trifluoroacetic acidand can be cleaved under a variety of conditions including, liquidhydrogen fluoride, aminolysis, hydrolysis, hydrazinolysis, catalytichydrogenation, or lithium borohydride to give a peptide acid, amide,hydrazide, or primary alcohol (Stewart and Young (1984) in Solid PhasePeptide Synthesis, sec. ed., Pierce Chemical Company, Illinois pp.88-95).

Netropsin and Distamycin A (FIGS. 2A and 2B) are heterocyclicpolyamides, containing imidazole (Im) and pyrrole (Py) carboxamides.These compounds are isolated from Streptomyces distallicus and exhibitantibiotic, antiviral and antitumor activity. Other members of thisfamily of antibiotics include noformycin (Diana (1973) J. Med. Chem.16:3774-3779), kikumycin B (Takaishi et al. (1972) Tetrahedron Lett.1873), and anthelvencin (Probst et al. (1965) Antimicrob. AgentsChemother. 789). Netropsin and Distamycin A are two examples of the manysmall molecules (MW<2 kD) which can bind and/or cleave DNA with modestsequence specificity (Krugh (1994) Curr. Opin. Struct. 4:351-364). Thesedrugs block template function by binding to specific nucleotides in theminor groove of double-stranded DNA.

Due to the pharmaceutical potential of this family of peptides aconsiderable amount of research has been devoted to the study of thesecompounds and their analogues. The x-ray crystal structure of a 1:1complex of Netropsin with the B-DNA dodecamer 5'-CGCGAATTCGCG-3' (SEQ IDNO:1) provides an understanding of how the sequence specificity isachieved, revealing that the amide hydrogens of theN-methylpyrrolecarboxamides form bifurcated hydrogen bonds with adenineN3 and thymidine O2 atoms on the floor of the minor groove. (Koopka etal. (1985) Proc. Natl. Acad. Sci. 82:1376; Koopka et al. (1985) J. Mol.Biol. 183:553). The pyrrole rings completely fill the groove excludingthe guanine amino group of a G, C base pair while making extensive vander Waals contacts with the walls of the groove, thereby affordingspecificity for A,T sequences. (Taylor et al. (1985) Tetrahedron 40:457;Schultz and Dervan (1984) J. Biomol. Struct. Dyn. 1:1133). Efforts todesign ligands specific for G, C containing sequences, were largelyunsuccessful (see e.g., Lown et al. (1986) Biochemistry 25:7408;Kssinger et al. (1987) Biochemistry 26:5590; Lee et al. (1987)Biochemistry 27:445; Lee et al. (1993) Biochemistry 32:4237), until thediscovery that two polyamides combine side-by-side in the minor grooveof DNA, forming a 2:1 complex with the DNA. (Pelton (1989) Proc. Natl.Acad. Sci., USA 86:5723-5727; Pelton (1990) J. Am. Chem. Soc.112:1393-1399; Chen et al. (1994) M. Struct. Biol. Nat. 1:169-175; Wadeet al. (1992) J. Am. Chem. Soc. 114:8783-8794; Mrksich et al. (1992)Proc. Natl. Acad. Sci., USA 89:7586-7590; Wade (1993) Biochemistry32:11385-11389; Mrksich et al. (1994) J. Am. Chem. Soc. 116:7983-7988).Each ligand interacts with one of the DNA strands in the minor groove,with the imidazole nitrogen making specific hydrogen bonds with oneguanine amino group. Thus, both Distamycin A and imidazole containingligands such as the designed polyamideimidazole-pyrrole-pyrrole-dimethylaminoproplyamine (ImPyPy-Dp),1-methylimidazole-2-carboxamide Netropsin, bind specifically in theminor groove as 2:1 polyamide/DNA complexes recognizing G, C sequences.

From studies of the 2:1 model it is now known that the combination ofimidazole/pyrrole carboxamide recognize a G, C base pair, and thecombination of pyrrole carboxamide/imidazole recognizes a C, G basepair, the pyrrole carboxamide/pyrrole carboxamide combination ispartially degenerate for T, A and A, T. The utility of the 2:1 model asan aid in designing ligands with sequence specificity for DNA isillustrated by the designed polyamideimidazole-pyrrole-imidazole-pyrrole-dimethylaminopropylamine(ImPyImPy-Dp) which binds a four base pair core sequence 5'-GCGC-3'.This is a complete reversal of the natural specificity of Netropsin andDistamycin A.

The literature contains a number of reports of the total synthesis ofvarious members of this family of polyamides and their analogues. All ofthe reported syntheses have been performed in the solution phase. Theamide bond unit in these polyamides is formed from an aromaticcarboxylic acid and an aromatic amine, both of which have provenproblematic for solution phase coupling reactions. The aromatic acidsare often unstable resulting in decarboxylation and the aromatic amineshave been found to be highly air and light sensitive (Lown and Krowicki(1985) J. Org. Chem. 50:3774-3779). It was believed that the variablecoupling yields, long (often >24 hour) reaction times, numerous sideproducts, and wide scale use of acid chloride and trichloroketoneintermediates in solution phase coupling reactions would make thesynthesis of the aromatic carboxamides difficult, if not impossible bysolid phase methods (He et al. (1993) J. Am. Chem. Soc. 115:7061-7071;Church et al. (1990) Biochemistry 29:6827-6838; Nishiwaki et al. (1988)Heterocycles 27:1945-1952). Thus, to date, there have been no reportedattempts to synthesize this class of compounds using solid phasemethodology.

The process of developing new ligands with novel sequence specificitygenerally involves four stages; design, synthesis, testing, and redesignof the model (Dervan (1986) Science 232:464). While exploring the limitsof the 2:1 model, the synthetic portion of the process emerged as themajor limiting factor, especially when confronted with expanding the 2:1motif to include longer sequences recognized by increasingly largerpolyamides. For example, the total synthesis of hairpin octa-amides suchas AcImImPy-γ-PyPyPy-G-Dp and AcPyPyPy-γ-ImImPy-G-Dp (FIGS. 3A and 3B)is characterized by difficult purifications. (γ representsγ-aminobutyric acid and G represents guanine.) Each polyamide wouldlikely require more than a months effort, even in the hands of a skilledresearcher. Methods for expediting the synthesis of analogs ofDistamycin A were investigated and the present invention describes anovel method for the synthesis of oligopeptides containing imidazole andpyrrole carboxamides on a solid support.

Oligonucleotide-directed triple helix formation is one of the mosteffective methods for accomplishing the sequence specific recognition ofdouble helical DNA. (See e.g., Moser and Dervan (1987) Science 238:645;Le Doan et al. (1987) Nucleic Acids Res. 15:7749; Maher et al. (1989)Science 245:725; Beal and Dervan (1991) Science 251:1360; Strobel et al.(1991) Science 254:1639; Maher et al. (1992) Biochemistry 31:70). Triplehelices form as the result of hydrogen bonding between bases in a thirdstrand of DNA and duplex base pairs in the double stranded DNA, viaHoogsteen base pairs. Pyrimidine rich oligonucleotides bind specificallyto purine tracts in the major groove of double helical DNA parallel tothe Watson-Crick (W-C) purine strand (Moser and Dervan (1987) Science238:645). Specificity is derived from thymine (T) recognition ofadenine-thymine base pairs (T→AT) base triplets and protonated cytosine(C⁺) recognition of guanine-cytosine base pairs (C⁺ →GC). (Felsenfeld etal. (1957) J. Am. Chem. Soc. 79:2023; Howard et al. (1964) Biochem.Biophys. Res. Commun. 17:93; Rajagopal and Feigon (1989) Nature 339:637;Radhakrishnan et al. (1991) Biochemistry 30:9022). Purine-richoligonucleotides, on the other hand, bind in the major groove of purinerich tracts of double helical DNA antiparallel to the W-C purine strand.(Beal and Dervan (1991) Science 251:1360). Specificity is derived fromguanine recognition of GC base pairs (G→GC base triplets) and adeninerecognition of AT base pairs (A→AT base triplets). (Durland et al.(1991) Biochemistry 30:9246; Pilch et al. (1991) Biochemistry 30:6081;Radhakrishnan et al. (1991) J. Mol. Biol. 221:1403; Beal and Dervan(1992) Nucleic Acids Res. 20: 2773). Oligonucleotide directed triplehelix formation is therefore limited mainly to purine tracts.

A major challenge in the sequence specific recognition of duplex DNA bytriple helix formation is designing oligonucleotides capable of bindingall four base pairs. Efforts toward this goal have included the designof non-natural heterocycles for the completion of the triplex code andthe design of oligonucleotides capable of binding alternate strands ofduplex DNA by triple-helix formation. (Beal and Dervan (1992) J. Am.Chem. Soc. 114:4976-4982; Stiltz and Dervan (1992) Biochem. 9:2177-2185;Koshlap et al. (1993) J. Am. Chem. Soc. 115:7908-7909).

An increasingly versatile method for accomplishing the sequence specificrecognition of DNA is the use of natural DNA binding molecules withaltered sequence specificity. (Dervan (1986) Science 232:464). Theconstruction of oligonucleotide-minor groove polyamide conjugates, usingnatural DNA binding molecules, such as Netropsin and Distamycin A,offers a promising method for expanding the number of sequences whichcan be targeted by oligonucleotide directed triple helix formation.

A number of methods have been reported for the synthesis of commonoligonucleotide-polyamide conjugates, based on post-syntheticmodification (Ede et al. (1994) Bioconj. Chem. 5:373-378; Haralambidiset al. (1993) Bioorg. and Med. Chem. Let. 4:1005-1010); assembly of apeptide on controlled pore glass followed by oligonucleotide synthesis(Haralambidis et al. (1990) Nuc. Acid. Res. 18:493-499; Haralambidis etal. (1987) Tet. Lett. 28:5199-5202; Tong et al. (1993) J. Org. Chem.58:2223-2231; Tung et al. (1991) Bioconj. Chem. 2:464-465; Bongratz etal. (1994) Nuc. Acid. Res. 22:4681-4688; Zhu and Stein (1994) Bioconj.Chem. 5:312-315) and synthesis of amino modified oligonucleotidesfollowed by solid phase synthesis of peptides.

There are a number of conceivable approaches to the design ofoligonucleotide-polyamide conjugates capable of recognizing doublehelical DNA by triple helix formation. In one approach, the conjugatecan be designed such that two minor-groove polyamide oligonucleotideconjugates bind antiparallel to a sequence of duplex DNA, with bindingmediated by the dimerization of the individual polyamide moieties in theminor groove of DNA, FIG. 20A. In a second approach, the conjugate canbe designed such that a single oligonucleotide head-to-tail hairpinpolyamide dimer, binds a sequence of duplex DNA in the minor groove,with binding mediated by oligonucleotide directed triple helix formationin the major groove, FIG. 20B. In each of these designs specificity isderived from specific contacts in the major groove from the pyrimidinemotif triple helix and in the minor groove from the 2:1 polyamide:DNAcomplex.

BRIEF SUMMARY OF THE INVENTION

The present invention describes a novel method for the preparation ofacyclic polyamides containing imidazole and pyrrole carboxamides. Thepresent invention also describes a novel method for the synthesis ofcyclic polyamides containing imidazole and pyrrole carboxamides. Furtherincluded in the present invention is a novel method for the solid phasesynthesis of imidazole and pyrrole polyamide-oligonucleotide andpolyamide-protein conjugates capable of recognizing double stranded DNA.

Included in the present invention is the solid phase synthesis ofanalogs of the di- and tri-N-methylpyrrolecarboxamide antiviralantibiotics Netropsin and Distamycin A. (FIGS. 2B and 2B).

This invention includes reaction schemes for producing a wide variety ofimidazole and pyrrole polyamides and imidazole and pyrrolepolyamide-oligonucleotide and protein conjugates. A key element in thesynthesis of these compounds is the use of a solid support inconjunction with Boc-(Boc=tert-butoxycarbonyl) andFmoc-(Fmoc=9-fluorenylmethyl carbonyl) chemistry.

More specifically, the invention provides a method for the solid phasesynthesis of imidazole and pyrrole polyamides comprising the steps of:preparing a solid support, preferably a polystyrene resin, forattachment of the polyamide to be synthesized; protecting and activatingthe appropriate amino acid monomers or dimers; sequentially adding theamino acid monomers or dimers to the solid support beginning with thecarboxy terminal amino acid; deprotecting the amino acids afterformation of the desired polyamide; cleaving the polyamide from thesolid support and purifying the synthesized polyamide.

Further included in the present invention are novel amino acid monomersand dimers and novel methods for synthesizing the same.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the basic approach for solid phase peptide synthesis.

FIG. 2A depicts the di-N-methylpyrrole-carboxamide antibiotic Netropsin.

FIG. 2B depicts the tri-N-methylpyrrole-carboxamide antibioticDistamycin A.

FIG. 3 depicts octapeptide hairpin dimers AcImImy-γ-PyPyPy-G-Dp (FIG.3A) and AcPyPyPy-γ-ImImPy-G-Dp (FIG. 3B), which are designed to bind thesites 5'-WGGWW-3' and 5'-WGGWW-3', respectively. W is either an A,T orT,A base pair.

FIGS. 4A-4D illustrate C3-methyl pyrrole inhibition of pyrrolecarboxamide-pyrrole carboxamide recognition of a GC base pair. Thepolyamide oligonucleotide complexes depicted in the Figures areImPyPy-γ-PyPyPy-Dp·TGTTA and ImPyPy-γ-PyPyPy-Dp·TGTCA. In FIGS. 4A and4B the polyamide is unmodified and in FIGS. 4C and 4D the third pyrroleof the polyamide has been modified by addition of a methyl group to thesecond carbon of the pyrrole ring.

FIG. 5 illustrates the potential for the formation of a bifurcatedhydrogen bond between a 3-substituted hydroxypyrrole carboxamide and thecarbonyl of thymine (dR represents deoxyribose).

FIG. 6A illustrates the additional hydrogen bond which can form betweenthe hydroxyl group of a 3-substituted hydroxypyrrole carboxamide and thecarbonyl of thymine.

FIG. 6B illustrates that adenine cannot form this additional hydrogenbond.

FIG. 7 depicts illustrative polyamides prepared by the method of thisinvention.

FIG. 8 illustrates a typical 72 minute solid phase synthesis cycle forminor groove polyamides according to one embodiment of this invention.

FIG. 9 illustrates three representative analytical high pressure liquidchromatography (HPLC) traces for stepwise monitoring of a solid phasepolyamide synthesis cycle from the synthesis of AcImImPy-γ-PyPyPy-G-Dp(0.1% wt/v TFA, gradient elution 1.25% CH₃ CN/min monitored at 254 nm).FIG. 9A depicts the HPLC spectrum of Boc-Py-γ-PyPyPy-G-Dp(Boc=tert-butoxycarbonyl) which elutes at 31.6 minutes. FIG. 9B depictsthe spectrum of H₂ N-Py-γ-PyPyPy-G-Dp which elutes at 24.3 minutes andFIG. 9C depicts the spectrum of Boc-ImPy-γ-PyPyPy-G-Dp which elutes at31.8 minutes.

FIG. 10 illustrates various spectra of the HPLC purified polyamide,AcImImPy-γ-PyPyPy-G-Dp (4a) (Scheme 2). FIG. 10A depicts the HPLCspectrum (0.1% wt/v TFA, 1.25% CH₃ CN/min), monitored at 254 nm. FIG.10B depicts the MALDI-TOF mass spectrum, internal standard at 1802.1 (M⁺H calculated for C₄₇ H₆₀ N₁₈ O₉, 1022.1, found 1022.4). FIG. 10C depictsthe ¹ H NMR spectra recorded at 300 MHz in d₆ -DMSO.

FIG. 11 depicts ribbon models of "slipped" (11A) and "overlapped" (11B)2:1 polyamide:DNA complexes.

FIGS. 12A-12H depict graphically the data obtained from the quantitativeDNase I footprint titration experiments. The (θ_(norm), [L]_(tot)) datapoints were obtained as described in Example 10.

FIG. 13 depicts the structures of the C-termini of various polyamidesillustrating that polyamides with C-termini (a) Py-Dp and (b) Py-β-Dp(β=β-alanine) sequences present similar surfaces to the minor groove,while polyamides with C-termini (c) Py-G-Dp sequences present adifferent surface. In the case of (c) the glycine carbonyl group isdirected toward the minor groove.

FIG. 14 illustrates the predicted 2:1 complexes of ImPyPy-X-PyPyPy-G-Dp,where X=G, β or Py, with the targeted sites (a) 5'-AAAAAGACAAAAA-3' (SEQID NO:2), (b) 5'-ATATAGACATATA-3' (SEQ ID NO:3) (13 bp, "slipped") and(c) 5'-TGTTAAACA-3' (SEQ ID NO:4) (9 bp, "overlapped"). The shaded andlight circles represent imidazole and pyrrole rings, respectively, andthe diamond represents the internal amino acid X. The specificallytargeted guanines are highlighted.

FIGS. 15A-15H depict the storage phosphor autoradiograms of the 8%denaturing polyacrylamide gels used to separate the fragments generatedby DNase I digestion in the quantitative footprint titration experiments(Example 10). The five binding sites analyzed in the footprint titrationexperiments are indicated on the right sides of the autoradiogram.

FIG. 16 depicts graphically the measurement of the time of the couplingof Boc-Py-OBt to PyNH₂ by picric acid titration. Samples were taken atone minute intervals.

FIG. 17 illustrates two ways in which double helical DNA can berecognized by an oligoncleotide-minor groove binding polyamideconjugate. In FIG. 17A oligonucleotide directed triple helix formationin the major groove is mediated by polyamide dimerization in the minorgroove. In FIG. 17B directed binding of a head-to-tail polyamide dimerin the minor groove is mediated by oligonucleotide directed triple helixformation in the major groove.

FIG. 18 depicts illustrative polyamide-oligonucleotide conjugatessynthesized by the method of this invention. Included in the figure arethe calculated and observed masses (MALDI-TOF).

FIG. 19 depicts a ribbon graphic illustrating how theoligonucleotide-polyamide conjugateDp-G-PyPyPy-G-PyPyIm-linker-TTTTTT^(m) C^(m) CTTT might bind to doublehelical DNA.

FIG. 20 illustrates a ribbon model of the GCN-4 protein's coiled and DNAbinding region binding to DNA.

FIG. 21 depicts a ribbon model of the GCN-4-polyamide conjugateillustrating the binding region of the substituted polyamide to DNA.

FIG. 22 depicts a schematic representation of a peptide synthesizer.

FIG. 23 depicts a flow chart of the computer program used to producepolyamides in the peptide synthesizer illustrated in FIG. 22.

FIG. 24 depicts a ball and stick model of the projected binding mode ofpolyamide H₂ N-β-PyPyPy-γ-ImImPy-β-β-β-β-PyPyPy-γ-ImImPy-β-Dp with thetarget DNA sequence 5'-TGGTTAGTACCT-3' (SEQ ID NO:5).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for the solid phase synthesis ofstraight chain and cyclic polyamides containing imidazole and pyrrolecarboxamides. The present invention also provides a method for the solidphase synthesis of imidazole and pyrrole polyamide-oligonucleotideconjugates and imidazole and pyrrole polyamide-protein conjugates.

Included in the present invention is the solid phase synthesis ofanalogs of the di- and tri-N-methylpyrrolecarboxamide antibioticsNetropsin and Distamycin A (FIGS. 2A and 2B).

More specifically the present invention includes a method for thepreparation and identification of pyrrole and imidazole carboxamidepolyamides and polyamide-oligonucleotide and protein conjugates whichrecognize double stranded DNA by interaction with the minor groove ofthe DNA.

Illustrative imidazole and pyrrole polyamides produced by the method ofthis invention are shown in FIG. 7 and Tables 1 and 4. Illustrativeimidazole and pyrrole carboxamide polyamide-oligonucleotide conjugatesproduced by the method of this invention are shown in Table 5 and FIG.18.

The present invention extends to all novel imidazole and pyrrolecarboxamide polyamides, imidazole and pyrrole carboxamidepolyamide-oligonucleotide conjugates and imidazole and pyrrolecarboxamide polyamide-protein conjugates that can be prepared accordingto the methods of the present invention.

Further included in this invention is an improved method for thepreparation of the Boc-pyrrole-OBt and Boc-imidazole-OBt activated aminoacid monomers and a novel method for the preparation of theFmoc-pyrrole-OBt and Fmoc-imidazole-OBt activated amino acid monomers.Also included in this invention are novel monomers in which the pyrroleis substituted at the N-methyl position and at the 3 position of thepyrrole ring. Also included is a novel method for the preparation ofimidazole containing dimers.

Certain terms used to describe the invention herein are defined asfollows:

The term "polyamide" is used to describe the polypeptides synthesized bythe method of this invention. A polyamide is a polymer of amino acidschemically bound by amide linkages (CONH). An "amino acid" is defined asan organic molecule containing both an amino group (NH₂) and acarboxylic acid (COOH). The polyamides of this invention are comprisedof imidazole carboxamides, pyrrole carboxamides, aliphatic amino acids,aromatic amino acids and any chemical modifications thereof.

The term "amino acid monomer" refers to a pyrrole or imidazole aminoacid or an aliphatic or aromatic amino acid in which the amine has beenprotected with the Boc-protecting group, Fmoc-protecting group orallyl-protecting group and the carboxylic acid has been activated as the-OBt ester or the symmetric anhydride.

The activated "pyrrole amino acid monomers" of this invention aregenerally depicted as follows: ##STR1## wherein R is a protecting groupindependently selected from the groups consisting of tert-butoxycarbonyl(Boc-), allyl (--CH₂ CH═CH₂) or 9-fluoroenylmethyl carbonyl (Fmoc-); R₁is independently selected from the group consisting of H, CH₃, OH, NH₂,Cl or CF₃ ; and R₂ is independently selected from the group consistingof H, C1-C10 alkyl, such as methyl, ethyl or isopropyl, C1-C10 alkenyl,C1-C10 alkynyl, such as --C.tbd.CCH₃, or a carboxylic acid, such as--CH₂ COOH.

The activated "imidazole amino acid monomers" of this invention aregenerally depicted as follows: ##STR2## wherein R is a protecting groupindependently selected from the groups consisting of tert-butoxycarbonyl(Boc-), allyl (--CH₂ CH═CH₂) or 9-fluoroenylmethylcarbonyl (Fmoc-).

The -OBt activated "dimers" of this invention are generally depicted asfollows: ##STR3## wherein R₃ is independently selected from the groupconsisting of an aliphatic or aromatic amino acid, an imidazolecarboxamide or a pyrrole carboxamide or any chemical modificationthereof.

"Oligonucleotide-polyamide conjugate" is a term used to describe amolecule which is comprised of an oligonucleotide chain and a polyamidechain joined by a covalent linkage.

"Protein-polyamide conjugate" is a term used to describe a moleculewhich is comprised of a protein chain and a polyamide chain joined by acovalent linkage.

"Nucleoside" means either a deoxyribonucleoside or a ribonucleoside orany chemical modifications thereof. Modifications of the nucleosidesinclude, but are not limited to, 2'-position ribose modifications,5-position pyrimidine modifications, 8-position purine modifications,modifications at cytosine exocyclic amines and the like.

A "failure sequence" refers to a polyamide chain which has not reactedwith the pyrrole/imidazole monomer or dimer during a particular reactioncycle.

The solid phase polyamide synthesis protocols of this invention weremodified from the in situ neutralization, Boc-chemistry recentlyreported by Kent and coworkers (Schnolzer et al. (1992) Int. J. PeptideProtein Res. 40:180-193; Milton et al. (1992) Science 252:1445-1448). Inits most basic form the method of preparing imidazole and pyrrolecarboxamide polyamides according to the present invention may be definedby the following series of steps: (1) The solid support, preferably apolystyrene resin, is prepared. The polystyrene resin is prepared byreaction with a linker molecule to enable facile attachment and removalof the polyamide. In one embodiment a spacer molecule is attached to thepolyamide prior to attachment of the linker molecule. (2) Theappropriate amino acid (aa) monomer or dimer is then protected at theamino (NH₂) group and activated at the carboxylic acid (COOH) group. Theamino (NH₂) group is protected with a Boc-group an Fmoc-group and thecarboxylic acid is activated by the formation of the -OBt ester, togive, in the case of the pyrrole and imidazole amino acidsBoc-pyrrole-OBt (9), Boc-imidazole-OBt (13), Fmoc-pyrrole-OBt (21a) andFmoc-imidazole-OBt (21b). (3) The protected and activated amino acidsare then sequentially added to the solid support beginning with thecarboxy terminal amino acid. High concentrations of activated monomerresults in fast coupling reactions and in situ neutralization chemistryassures that the unstable deprotonated amine is generated simultaneouswith the initiation of a coupling reaction. Coupling times are rapid,generally 72 minutes per residue, and simple, requiring no specialprecautions beyond those required for ordinary solid phase peptidesynthesis. (4) When the desired polyamide has been prepared the aminoacids are deprotected and the peptide is cleaved from the resin andpurified. The reactions are periodically monitored using picric acidtitration and high pressure liquid chromatography (HPLC). Each of thesesteps are described in detail below. The synthesis ofImPyPyPyPyPyPy-G-ED (G=glycine, ED=ethylenediamine) 1a,ImPyPyPyPyPyPy-G-Dp (Dp=dimethylaminopropylamine) 1b,ImPyPyPyPyPyPy-G-Ta (Ta=3,3'-diamino-N-methylpropylamine) 1c,ImPyPyPyPyPyPy-G-Ta-EDTA (EDTA=ethylenediaminetetraacetic acid) 1d,ImPyPy-G-PyPyPy-G-ED 2a, ImPyPy-G-PyPyPy-Dp 2b, AcImPyPy-G-PyPyPy-G-Dp(Ac=acyl) 2c, AcImPyPy-G-PyPyPy-G-Ta-EDTA 2d, AcImPyPy-γ-PyPyPy-G-Dp 3a,AcImPyPy-γ-PyPyPy-G-Ta 3b, AcImPyPy-γ-PyPyPy-G-EDTA 3c,AcImImPy-γ-PyPyPy-G-Dp 4a, AcImImPy-γ-PyPyPy-G-Ta 4b,AcImImPy-γ-PyPyPy-G-EDTA 4c, AcPyPyPy-γ-ImImPy-G-Dp 5a,AcPyPyPy-γ-ImImPy-G-Ta 5b, and AcPyPyPy-γ-ImImPy-G-Ta-EDTA 5c (FIG. 7)is described herein. A complete list of illustrative polyamidessynthesized by the methods of this invention is set forth in Table 1.All compounds listed in this table have been characterized by ¹ H NMR,HPLC, MALDI-TOF mass spectroscopy and in some cases ¹³ C NMR.

The pyrrole and imidazole polyamides of this invention are contemplatedfor use as antiviral, antibacterial and antitumor compounds whichrecognize double stranded DNA by interaction with the minor groove ofthe DNA. Specifically, it is anticipated that the pyrrole and imidazolepolyamides may be used to sequence DNA ligands which are able tospecifically inhibit DNA binding proteins, such as transcription factorswhich are responsible for gene regulation, thus, providing a basis forrapid rational design of therapeutic compounds. Theethylenediaminetetraacetic acid (EDTA) derivatives of the polyamidessynthesized by the method of this invention are also contemplated foruse in the field of molecular biology. These molecules can be used tobind and cleave double stranded DNA at a specific site using iron (Fe)and EDTA.

It is further contemplated that the novel N-substituted pyrrole monomersof this invention will provide polyamides with novel DNA bindingproperties, with enhanced pharmacological properties, or providefunctionalized polyamides for synthesis of modified derivatives.

In its most basic form the method of preparing imidazole and pyrrolepolyamide-oligonucleotide conjugates according to the present inventionmay be defined by the following series of steps: (1) The oligonucleotideis assembled on a solid support using standard methodology. (2) Theappropriate amino acid monomer is then protected and activated. Theamino group is protected with the Boc- or Fmoc-group and the aromaticacid is activated by the formation of the -OBt ester, to give, in thecase of the pyrrole and imidazole amino acids, Boc-pyrrole-OBt (9) andBoc-imidazole-OBt (13) Fmoc-pyrrole-OBt (21a) and Fmoc-imidazole-OBt(21b). (3) The protected and activated amino acids are then sequentiallyadded to the assembled oligonucleotide beginning with thecarboxyterminal amino acid. As stated above, high concentrations ofactivated monomer results in fast coupling reactions and in situneutralization chemistry assures that the unstable deprotonated amine isgenerated simultaneous with the initiation of a coupling reaction.Coupling times are rapid, generally 72 minutes per residue, and simple,requiring no special precautions beyond those required for ordinarysolid phase peptide synthesis. (4) When the desired polyamide has beenprepared the amino acids are deprotected and the polyamide-conjugate iscleaved from the resin.

The pyrrole and imidazole polyamide-oligonucleotide conjugates of thisinvention are contemplated for use as potential antiviral compoundswhich recognize double stranded DNA by triple helix formation. ManyDNA-binding proteins bind in the major groove of DNA. It is anticipatedthat polyamide-oligonucleotide conjugates may be more effectiveinhibitors of sequence specific DNA binding proteins, since they willocclude both the major and minor grooves.

In its most basic form the method of preparing imidazole and pyrrolepolyamide-protein conjugates according to the present invention may bedefined by the following series of steps: (1) The protein is assembledusing standard methodology; (2) The appropriate amino acid monomer isthen protected and activated as discussed above. (3) The protected andactivated amino acid monomers are then sequentially added to theassembled protein beginning with the carboxyterminal amino acid. (4)When the desired polyamide has been prepared the amino acids aredeprotected and the polyamide-conjugate is cleaved from the resin.

The pyrrole and imidazole polyamide-protein conjugates of this inventionare contemplated for use as potential antiviral, antibacterial andantitumor compounds which recognize double stranded DNA by interactionwith the minor groove of DNA. Many DNA-binding proteins bind in themajor groove of DNA. It is anticipated that the appended peptide moietywill provide a means for introducing the polyamide into the cell.

Synthesis of the Imidazole and Pyrrole Amino Acid Monomers

The Boc-pyrrole-OBt (Boc-Py-OBt) (9) and Boc-imidazole-OBt (Boc-Im-OBt)(13) monomers are synthesized using a modified procedure of Grehn andcoworkers (Grehn and Ragnarsson (1991) J. Org. Chem. 46:3492-3497; Grehnet al. (1990) Acta. Chem. Scand. 44:67-74) (Example 1 and Scheme 9). Themodification involves the use of the commercially availableBoc-anhydride (di-t-butyl-dicarbonate) as the Bocing agent, rather thanthe highly reactive Bocing agent, tert-butyloxycarbonyl fluoride(Boc-fluoride) employed by Grehn. Boc-fluoride is dangerous to preparein large quantities, requiring the use of chlorofluorophosgene which isvery toxic. Additionally, Boc-fluoride is not stable for storage(Wackerle and Ugi (1975) Synthesis 598-599; Franzen and Ragnarsson(1979) Acta. Chem. Scand. 33:690-692; Dang and Olofson (1990) J. Org.Chem. 55:1847-1851). The reaction of Boc-anhydride with the pyrroleamino group has been reported by Bailey et al. (1989) J. Pharm. Sci. 78:910-917. Overall yields starting from the nitro/methyl esters arereproducibly greater than 60% for both the pyrrole and imidazole -OBtesters, with simple purification requiring no column chromatography.Additionally, the Boc-imidazole-OBt ester prepared by this procedure isstable at room temperature.

The Fmoc-protected monomers are synthesized from the Boc-protectedmonomers as illustrated in Scheme 12 (Example 1). Fmoc- is an alternateprotecting group commonly used for peptide synthesis. Fmoc- is removedwith dilute base, whereas the Boc-group is removed under acidicconditions. The use of Fmoc- as a protecting group provides additionalversatility to the method of this invention.

Boc-pyrrole monomers substituted at the N-methyl position having thefollowing general formula: ##STR4## where R₂ is independently selectedfrom the group consisting of H, C1-C10 alkyl, such as methyl, ethyl orisopentyl, a 1° or 2° amine, such as N, N, dimethylpropylamine,ethylamine, a carboxylic acid, such as --CH₂ --COOH, an alkenyl, or analkynyl, such as --.tbd.--CH₃ are prepared as illustrated in Scheme 1.As stated above, it is believed that such compounds will providepolyamides with enhanced pharmacological properties. A general methodfor the preparation of these compounds is as follows: ##STR5## (SeeExample 1, Scheme 13).

Pyrrole monomers substituted at the 3 position of the pyrrole ringhaving the following general formula: ##STR6## wherein R₁ isindependently selected from the group consisting of a C1-C10 alkylgroup, such as --CH₃, or an --OH, --NH₂, or --OR₄, wherein R₄ is aC1-C10 alkyl group, such as methyl or allyl and R₂ is independentlyselected from the group consisting of H, C1-C10 alkyl, such as methyl,ethyl or isopentyl, a 1° or 2° amine, such as N, N, dimethylpropylamine,ethylamine, a carboxylic acid, such as --CH₂ --COOH, an alkenyl, or analkynyl, such as --.tbd.--CH₃, were synthesized as discussed in Example1 (Schemes 14 and 15). Such compounds are of interest because they allowsubstitution of a proton which is known by structural studies to betightly associated in the floor of the minor groove (see FIG. 4). Asdiscussed below, monomers substituted at the 3 position of the pyrrolering will likely provide polyamides with novel DNA binding properties.

While studying polyamide DNA complexes, it was discovered that therecognition of a GC base pair by the combination of a pyrrolecarboxamide opposite a pyrrole carboxamide is a potential mismatch.(FIG. 4C) (Mirksich et al. (1994) J. Am. Chem. Soc. 116:6873-7988). Apolyamide prepared using a pyrrole monomer substituted at the 3 positionwith an alkyl group, therefore, will likely introduce a steric clashwhen pyrrole carboxamide/pyrrole carboxamide binds opposite GC. (FIG.4D). A,T recognition by the methyl derivative will not be greatlyinhibited. (FIG. 4B). A general method for the preparation of a pyrrolewith an alkyl substituent in the 3 position is set forth in Example 1(Scheme 14).

Substituted monomers in which R₁ is --OH or --NH₂ may be used tointroduce a specific hydrogen bond interaction between the --OH or --NH₂group of the pyrrole and the carbonyl of thymine, which is capable offorming bifurcated hydrogen bonds, and should be able to hydrogen bondto both a pyrrole carboxamide and the substituted pyrrole. Adenine, onthe other hand, will not be able to form this additional hydrogen bond,since it has only a single lone pair electron which is already hydrogenbonded to a pyrrole carboxamide. (See FIGS. 5 and 6). A general methodfor the preparation of a pyrrole with a hydroxy or alkoxy substituent inthe 3 position of the ring is set forth in Example 1 (Scheme 15).

Synthesis of Resin Linkage Agents

For solid phase synthesis, the growing polymer chain must be attached tothe insoluble matrix by a linkage agent which is stable for the courseof the synthesis, but cleaved in high yields under appropriateconditions to release the synthesized polymer. A number of resinlinkages were investigated. Of these, Merrifield's PAM(tert-butyloxycarbonylaminoacyl-pyrrole-4-(oxymethyl)phenyl-acetamidomethylresin (Boc-Py-PAM-resin), synthesized as described below, is thepreferred solid support because it is stable to trifluoroacetic acid(TFA) and because it can be cleaved from the resin under a varietyconditions including, liquid hydrogen fluoride, amminolysis, hydrolysis,hydrazinolysis, catalytic hydrogenation, or lithium borohydride to givea peptide acid, amide, hydrazide, or primary alcohol, as discussedabove. Two resin linkage agents, compounds 39 and 40 were employed.These compounds were prepared in three steps according to the publishedprocedures of Merrifield, using the Boc-protected pyrrole amino acid,Boc-Py-COOH (8) (Mitchell et al. (1978) J. Orga Chem. 43:2845-2852)(Example 3, Scheme 16). ##STR7## To attach the resin linkage agents tothe resin, compounds 39 and 40 were activated withdicyclohexylcarbodiimide (DCC) followed by reaction with aminomethylatedpolystyrene for 24 hours to give Boc-pyrrole-BAM-resin 41 andBoc-pyrrole-PAM-resin 42, respectively (Example 3). The reactions werepreferably stopped at 0.2 to 0.3 mmol/gram substitution as determined bythe quantitative ninhydrin test and by picric acid titration (Sarin etal. (1981) Anal. Biochem. 117:147-157; Gisin (1972) Anal. Chim. Acta.58:248). ##STR8## Activated resin linkage agents 39 and 40 were alsoreacted with the commercially available Boc-glycine-PAM-resin(Boc-G-PAM-resin) to give Boc-pyrrole-PAM-G-PAM-resin(Boc-Py-PAM-G-PAM-resin) and the corresponding BAM resin. Finally, thecommercially available Boc-G-PAM-resin and Boc-β-alanine-PAM-resin(Boc-β-PAM-resin) were reacted with Boc-Pyrrole-OBt (9) to yield theBoc-pyrrole-G-PAM-resin (43) (see Scheme 2, step a) and theBoc-pyrrole-β-PAM-resin (44), respectively. Unreacted amino groups werecapped by acetylation. ##STR9## The corresponding Boc-imidazole-G-PAMand Boc-imidazole-β-PAM-resins were synthesized using the sameprocedure.

Solid Phase Polyamide Synthesis Protocols

The solid phase peptide synthesis (SPPS) methods of this invention weremodified from the in situ neutralization chemistry described by Kent andcoworkers. (Schnolzer et al. (1992) Int. J. Peptide Protein Res.40:180-193; Milton et al. (1992) Science 252:1445-1448). One embodimentfor the synthesis of polyamides according to the method of thisinvention is listed in Table 2 and shown schematically in FIG. 8. (SeeExample 4). The Boc-protected monomers are used in Example 4 forpurposes of illustration. The methods illustrated are readily extendableto the Fmoc-protected monomers.

The method consists of washing either the Boc-pyrrole-PAM/BAM-resins (41and 42), or the Boc-pyrrole-G-PAM (43)/Boc-pyrrole-β-PAM (44) resins orthe corresponding Boc-imidazole resins with dichloromethane followed bythe removal of the Boc-group with 65% trifluoroacetic acid (TFA)/35%dichloromethane/0.5M thiophenol for 20 minutes. The deprotected resin isthen washed with dichloromethane followed by dimethylformamide (DMF). Asample of the resin can be analyzed at this time using the picric acidtest, as described below. After deprotection of the resin, an -OBtactivated amino acid monomer and diisopropylethylamine are added to theresin and the reaction is allowed to proceed for 45 minutes. After 45minutes a sample is taken for analysis and the resin is washed with DMF.The overall stepwise assembly of a single residue takes approximately 72minutes under the standard conditions.

The coupling of the -OBt ester of Boc-pyrrole to the imidazole amine onthe solid support was found to be slow using the above procedure. In apreferred embodiment of the invention, the pyrrole monomer was activatedas the symmetric anhydride, rather than the -OBt ester, using a modifiedprocedure of Ding et al. (1963) Acta Chem. Scand. 23:751. Ding describesthe solution phase coupling of pyrrole to pyrazole by formation of thesymmetric anhydride of the pyrrole monomer (DCC and DMAP indichloromethane) prior to coupling. This procedure was modified for useon a solid support and provides a rapid and effective method for thecoupling of pyrrole to imidazole. This modified procedure, which isdescribed in Example 5, provides a high yielding rapid reaction.Coupling yields were found to be greater than 98% for the synthesis ofthe polyamide AcPyImPy-G-Dp.

In developing a method for the solid phase synthesis of polyamides, itwas anticipated that intermolecular chain aggregation could be a severeproblem, with the adjacent extended aromatic polyamide providingexcellent surface areas for stacking. To minimize the possibility ofaggregation, a low substitution resin, 0.2 mmol or 0.3 mmol per gram, isused in combination with in situ neutralization. The high couplingyields (>99% in most cases), indicate that intermolecular interactionsare not a problem under these conditions. In situ neutralization hasalso been found to increase the lifetime of the aromatic amines.

In situ neutralization involves the elimination of a separateneutralization step by adding diisopropylethylamine (DIEA)simultaneously with the activated monomer. In standard solid phasepeptide synthesis it has been demonstrated by direct physicalmeasurement that intermolecular aggregation of the growing peptide chainis disrupted by TFA, which is an excellent solvent for most peptides,the aggregates reform, however, during the subsequent neutralization ofthe amine trifluoroacetate (Larsen et al. (1990) Peptides 183-185;Woerkom and Nipsen (1991) Int. J. Pep. Prot. Res. 38: 103-113; Milton etal. (1990) J. Am. Chem. Soc. 112:6039-6046). By adding the monomersimultaneously with neutralizing agent, coupling is able to occur beforean aggregate can form. In situ neutralization was adopted by Kent andcoworkers to eliminate low coupling yields resulting from intermolecularaggregation in Boc-chemistry SPPS. (Larson et al. (1990) Peptides183-185; Hudson (1988) J. Org. Chem. 53:617-624; Woerkom and Nipson(1991) Int. J. Pep. Prot. Res. 38:103-113).

In the standard in situ neutralization procedure DMF is used as thesolvent, because it maximizes the solvation of the growing peptidechain. Because the mixing of DMF and TFA is very exothermic the standardprocedure of Kent is modified by addition of the steps of washing theresin with dichloromethane both before and after treatment with TFA.Thiophenol is also added in the TFA deprotection step as a scavenger forthe t-butyl cation. This is necessitated by the potential for sidereactions between the t-butyl cation and the unprotected imidazolenitrogen, which has been reported to be nucleophilic in solution phasereactions. (Grehn and Ragnarsson (1981) J. Org. Chem. 46:3492-3497;Grehn et al. (1990) Acta. Chem. Scand. 44:67-74). Thiophenol, methylethyl sulfide, and ethanedithiol have all been reported to competeeffectively as scavengers of t-butyl cation. (Lundt et al. (1978) Int.J. Pep. Prot. Res. 12:258-268).

Monitoring the Progress of the Synthesis

In standard SPPS, the quantitative ninhydrin test is the preferredmethod of monitoring the coupling reactions and calculating yields.(Sarin et al. (1981) Anal. Biochem. 117:147-157; Gisin (1972) Anal.Chim. Acta. 58:248). The aromatic amines of pyrrole and imidazole,however, do not react in the quantitative ninhydrin test. In place ofthe ninhydrin test, picric acid titration and stepwise cleavage andmonitoring by HPLC are used to estimate coupling yields and monitor thecourse of the reactions. In the few cases where it is possible to usethe quantitative ninhydrin test, such as the coupling of Boc-Py-OBt toN₂ H-γ or H₂ N-G, all yields have been observed to be better than 99.8%.

Picric acid titration measures the amount of unreacted amine remaining.The method involves formation of the picrate salt of the amine, which isthen quantitated from the reported extinction coefficients. Theexperimental procedure is set forth in Example 6. The picric acid testis inaccurate for low concentrations of amine, thus it is only possibleto determine if a reaction is >90% complete using this measurement.Picric acid titration is useful for immediate monitoring of couplingreactions.

High pressure liquid chromatography (HPLC) is used for the stepwisemonitoring of the polyamide synthesis. After each coupling reaction asmall portion of resin is removed from the reaction mixture and thepolyamide is cleaved from the resin and analyzed by analytical HPLC, asdescribed in Example 7. The use of stepwise HPLC analysis is aneffective way to obtain detailed information on the progress of asynthesis, allowing the exact step that results in a side reaction ordeletion product to be readily identified and eliminated.

Cleavage of the Polyamide from the Boc-pyrrole-resin

Example 8 describes a general method for cleaving the polyamide from theBoc-Py-PAM/BAM-resins using Pd(OAc)₂. The successful cleavage of minorgroove polyamides is achieved from PAM and BAM pyrrole resins withPd(OAc)₂ in DMF under a pressurized atmosphere of hydrogen (100 psi, 8hours). Scheme 17 (Example 8) illustrates this procedure with theacetylated tripyrrole AcPyPyPy-PAM-resin. Upon being cleaved from theresin the terminal pyrrole acid can be activated withDCC/hydroxybenzotriazole (HOBt) and reacted with a primary amine toyield the corresponding amide.

Cleavage from PAM and BAM resins by amminolysis was unsuccessful at 37°C. and 60° C. in 1:1 amine:DMF or neat amine for 24 hours.

Cleavage of the Polyamide from the Boc-G-PAM-Resin

Example 9 describes a general method for the cleavage of the polyamidefrom the Boc-G-PAM-resin using a 1:1 mixture ofdimethylaminopropylamine:DMF. Scheme 18 (Example 9) illustrates thismethod with the acetylated tripyrrole AcPyPyPy-PAM-G-PAM-resin. Afterreaction with a 1:1 mixture of dimethylaminopropylamine:DMF at 37° C.for 12 hours, two products AcPyPyPy-PAM-G-Dp (98% of product) and thefailure sequence AcPyPy-PAM-G-Dp (2%) were identified by ¹ H NMR.Recovery of the product was very high--almost 50% of the theoreticalyield--indicating that the pyrrole-G-PAM-resin is cleaved with muchhigher recovery than the pyrrole-PAM-resin.

Based on the excellent recovery of acetylated tripyrrole underchemically mild conditions, the use of a glycine spacer is the preferredsynthetic method. This method offers two advantages, high cleavageyields from the resin and commercial availability of highly pureBoc-G-PAM-resin with 0.2 mmol/gram substitution.

Purification and Characterization of Peptides

Reversed phase HPLC purification provides a convenient and efficientmethod for the purification of the solid phase peptide products(Fransson et al. (1983) J. Chrom. 268:347-351). Amminolysis reactionsare filtered to remove the resin, diluted with water and immediatelypurified by reversed phase HPLC with a gradient of 0.15% CH₃ CN/min. in0.1% wt./v. TFA. A single preparatory run is sufficient to obtain puritygreater than 98% as determined by a combination of HPLC, ¹ H NMR andmass spectroscopy. ¹ H NMR is carried out in the non-exchangeablesolvent d₆ -DMSO, making all amide hydrogens and the trifluoroacetamideprotons clearly visible. 2-D COSY experiments are used to assist in theassignment of protons. MALDI-TOF mass spectroscopy provides an accurateand rapid method of confirming that full length product has beenisolated.

Scheme 2 illustrates a representative solid phase synthetic scheme forthe polyamide AcImImPy-γ-PyPyPy-G-Dp (Dp=dimethylaminopropylamine) (4a)starting from the commercially available Boc-G-PAM-resin. The polyamidewas synthesized with 7 standard synthesis cycles and the final productwas acetylated. The synthesis was monitored by analytical HPLC asillustrated in FIGS. 9A-9C. FIG. 9A, which depicts the spectrum ofBoc-Py-γ-PyPyPy-G-Dp, shows that the synthesis has proceeded after fivereaction cycles with a major peak eluting at 31.6 minutes observed. TheBoc-group is removed under standard conditions to give upon cleavage ofa small sample H₂ N-Py-γ-PyPyPy-G-PAM-resin which elutes at 24.3 minutes(FIG. 9B). The pyrrole amine is reacted with 4 equivalents of Boc-Im-OBtunder standard conditions, giving a quantitative conversion toBoc-ImPy-γ-PyPyPy-G-PAM-resin which is observed upon cleavage withdimethylaminopropylamine to elute at 31.8 minutes (FIG. 9C). Allcoupling reactions proceeded in greater than 90% yield as determined bypicric acid titration. The coupling of pyrrole to γ-aminobutyric acid(γ) and glycine proceeded in 99.9% yield as determined by thequantitative ninhydrin test. All yields are established as >99% by HPLCanalysis of each individual coupling step. Monitoring each individualstep before and after deprotection assures that high yields are beingobtained. Upon completion of the synthesis, the resin is cleaved byamminolysis with a 1:1 mixture of DMF and N,N-dimethylaminopropylamineat 37° C. for 12 hours. After 12 hours the reaction mixture is filteredto remove the resin, diluted with 4 volumes water and immediatelypurified by reversed phase HPLC with a gradient of 0. 15% CH₃ CN/min. in0.1% wt/v TFA. A single preparatory scale separation is sufficient toobtain purity greater than 98% as determined by a combination of HPLC, ¹H NMR and mass spectroscopy.

The HPLC, MS and ¹ H NMR spectra of the purified product are shown inFIGS. 10A-10C respectively. As can be seen in the ¹ H NMR spectrum (FIG.10C) only the expected protons are observed, from high field to lowfield, 2 imidazole carboxamide protons, four pyrrole carboxamideprotons, a trifluoroacetate proton (the tertiary amine is obtained asthe trifluoroacetate salt after HPLC purification in 0.1% TFA, and thetrifluoroacetate proton is identified by 2-D COSY experiments), thethree amides corresponding to the aliphatic amines, two imidazole ringprotons (singlets) and 8 pyrrole ring protons are observed as eitherdoublets or multiplets. MALDI-TOF mass spectroscopy (FIG. 10B) providesan accurate and rapid method of confirming that full length product hasbeen isolated and that side reactions such as alkylation or acylation ofthe unprotected imidazole nitrogen have not occurred. A combination ofanalyses ensures that pure full length peptide has been obtained in highpurity. ##STR10##

Synthesis of Polyamide Derivatives

The methods for the synthesis of minor-groove polyamides can be readilyextended to the synthesis of various derivatives. Scheme 3 illustratesthe synthetic scheme for introduction of EDTA into the C-terminus ofminor groove polyamides by cleavage from the resin with a symmetricaltriamine followed by reaction with EDTA monoanhydride. (See Example 4).##STR11## C-terminus EDTA derivatized polyamides are typically recoveredin approximately 30% yield after HPLC purification.

Example 4 further describes the synthesis of C-terminusdimethylaminoproplyamine (Dp), ethylenediamine (ED),3,3'-diamino-N-methylpropylamine (Ta) and β-alanine (β) derivatizedpolyamides. Finally, Example 4 also describes the synthesis ofN-terminus EDTA derivatized polyamides using the synthesis ofEDTA-γ-ImPyPy-β-PyPyPy-G-Dp as an example. Briefly, the polyamide H₂N-γ-ImPyPy-β-PyPyPy-G-Dp is prepared by cleavage of H₂N-γ-ImPyPy-β-PyPyPy-G-Resin with dimethylaminopropylamine (Dp). Theprimary amine is then derivatized with EDTA as described in Scheme IIIand isolated by preparatory HPLC.

The intermediates, containing a free primary amine, provide access to awide number of modified minor-groove polyamides, including, but notlimited to intercalators, polysaccharide conjugates, photoreactiveagents and metal chelates. Furthermore, a polyamide containing a freeprimary amine can be reacted with an activated carboxylic acid tosynthesize polyamides of increasing complexity, thiol modifiedpolyamides, or bromoacetic acid modified polyamides. Amine modifiedpolyamides are also useful for attachment to an appropriate support formaking affinity chromatography columns. The synthetic methods outlinedfor Boc-protected monomers substituted at the N-methyl group, allows forthe synthesis of amino modified pyrrole monomers for the addition ofEDTA into any region of the polyamide.

Effect of C-terminal Glycine and C-terminal β-alanine on DNA BindingProperties: Specifically for "Slipped" versus "Overlapped" Binding Modes

The DNA-binding affinities of several polyamides having the coresequence ImPyPy-X-PyPyPy (X=G, β, γ, Py) to the targeted 13 bp site5'-AAAAAGACAAAAA-3' (SEQ ID NO:2), and to the targeted 9 bp site5'-TGTTAAACA-3' (SEQ ID NO:4) were examined by DNase I footprinttitration (Galas and Schmitz (1978) Nucleic Acids Res. 5:3157-3170; Foxand Waring (1984) Nucleic Acids Res. 12:9271-9285; Brenowitz et al.(1986) Methods Enzymol. 130:132-181; Brenowitz et al. (1986) Proc. Natl.Acad Sci. U.S.A. 83:8462-8466; Senear et al. (1986) Biochemistry25:7344-7354) (Example 10, FIG. 15). The polyamide:DNA complexespredicted for the 13 bp and 9 bp target sites represent two distinctbinding modes, referred to as "slipped" and "overlapped" (see FIG. 11).The "slipped" (13 bp) binding mode (FIG. 11A) integrates the 2:1 and 1:1binding motifs at a single site. In this binding mode, the ImPyPyportion of two ImPyPy-X-PyPyPy polyamides bind the central 5'-AGACA-3'sequence in a 2:1 manner and the PyPyPy portion of the polyamides bindthe A/T flanking sequences similar to the 1:1 complexes of Distamycin.In the "overlapped" (9 bp) binding mode (FIG. 11B), two ImPyPy-X-PyPyPypolyamides bind directly opposite one another, with the ImPyPy portionof one polyamide opposite the PyPyPy portion of the other polyamiderecognizing the 5 bp subsides 5'-TGTTA-3' and 5'-AAACA-3' as in theImPyPy-Dp/Distamycin (PyPyPy) heterodimer.

In the 13 bp "slipped" and 9 bp "overlapped" sites described above, theGC and CG base-pairs are separated by one and five A/T base-pairs,respectively. It should be noted that "partially slipped" sites of 10,11 and 12 bp in which the GC and CG base-pairs are separated by two,three and four A/T base-pairs, respectively, are also potential bindingsites of the polyamides studied here.

Affinities for polyamides differing only in the presence or absence of aC-terminal glycine residue reveals that C-terminal glycine dramaticallyaffects the DNA-binding properties of polyamides. (See Table 3, FIG.12.) Relative to the polyamide ImPyPy-G-PyPyPy-Dp, which binds the"slipped" site 5'-AAAAAGACAAAAA-3' (SEQ ID NO:2) and the "overlapped"site 5'-TGTTAAACA-3' (SEQ ID NO:4) (with similar affinities(approximately 1×10⁸ M⁻¹) (FIG. 12A), polyamide ImPyPy-G-PyPyPy-G-Dp,which was previously prepared by solution methods and has a C-terminalglycine, binds these sites with approximately 1.5-fold (7×10⁷ M⁻¹) and80-fold (1.7×10⁶ M⁻¹) lower affinities, respectively (FIG. 12B). Also,relative to ImPyPy-G-PyPyPy-Dp, ImPyPy-G-PyPyPy-G-Dp binds to the 11 bpsite 5'-TGTGCTGCAAG-3' (SEQ ID NO:6) with >50-fold lower affinity (FIGS.12A and 12B). Each data point is the average value obtained from threequantitative footprint titration experiments (Example 10). Similarly,relative to ImPyPy-β-PyPyPy-Dp, ImPyPy-β-PyPyPy-G-Dp binds5'-AAAAAGACAAAAA-3' (SEQ ID NO:2) and 5'-TGTTAAACA-3' (SEQ ID NO:4) withapproximately 1.5-fold and 10-fold lower affinities, respectively (FIGS.12C and 12D). In both cases, C-terminal glycine confers specificity for"slipped" relative to "overlapped" complexes. In the case ofIrnPyPyPyPyPyPy-Dp and ImPyPyPyPyPyPy-G-Dp, the presence of a C-terminalglycine reduces the binding affinities at both the "slipped" and"overlapped" sites by factors of approximately 8 and 15, respectively(FIGS. 12G and 12H).

In contrast to ImPyPy-G-PyPyPy-Dp, ImPyPy-G-PyPyPy-β-Dp has DNA-bindingaffinities and specificities similar to ImPyPy-G-PyPyPy-Dp (Table 3,FIG. 12F). Modeling indicates that ImPyPy-G-PyPyPy-Dp andImPyPy-G-PyPyPy-β-Dp have similar DNA-binding surfaces at the C-terminalend of the polyamides (FIG. 13). The disruption of "overlapped" 2:1complexes by C-terminal glycine may result from a steric interactionbetween the glycine carbonyl group and the floor of the minor groove. Inthe "slipped" binding mode, the C-terminal part of the molecule is boundin a 1:1 manner, which is tolerant of C-terminal glycine (FIG. 14).

Rates and Efficiency of Coupling Reactions

Under the standard coupling conditions the efficiency of couplingreactions is as follows, (activated ester/free amine),Py/G≈Im/G>G/Py≈G/Im>Im/Py>Py/Py>Im/Im>Py/Im. All couplings except forIm/Im and Py/Im are >99.8% complete in 42 minutes. The faster couplingsare more than 99.8% complete within 5 minutes. For the Im/Im and Py/Imcouplings, extended reaction times are recommended in order to assurecomplete reaction. Fortunately, these couplings occur least frequentlyin the current synthesis of the minor groove polyamides. The Py/Pycoupling is the most common and was used as the model around which thesynthetic methodology was optimized. Coupling rates are estimated basedon picric acid titration data and ninhydrin tests when possible. Nocorrection was made for the change in substitution of the resinresulting from the addition of a monomer, because the effect is verysmall for the low substitution resins used for synthesis. The change insubstitution during a specific coupling or for the entire synthesis canbe calculated as

    L.sub.new =L.sub.old /(1+L.sub.old (W.sub.new -W.sub.old)×10.sup.-3)(1)

where L is the mmol of amine per gram of resin, and W is the weight(gmol⁻¹) of the growing polyamide attached to the resin. The subscriptold, indicates a parameter before the coupling reaction, new indicates aparameter referring to the resin after a coupling reaction.

For the Py/Py, Im/Py, Py/G and G/Py couplings an attempt was made tomeasure rates using picric acid titration at 1 minute time intervals.The Im/Py, Py/G, and G/Py couplings all reached completion too rapidlyto measure an accurate rate. For the Py/Py coupling, reasonably accuratedata was obtained for monitoring the disappearance of amine. From theslope of a plot of ln (meq. amine) versus time, it is possible toestimate a rate of reaction of 0.18h⁻¹ which corresponds to a 3.9 minutehalf life, and indicates that 25.6 minutes are required for 99%reaction, and 38.4 minutes for 99.9% reaction (FIG. 16). A 45 minutecoupling time was chosen to ensure complete reaction.

Preparation of Dimeric Building Blocks

As discussed above the amine group of imidazole is less reactive thanthe amine group of pyrrole. When coupling aliphatic amino acids to animidazole amine, extended coupling times or double coupling is sometimesrequired. For the coupling of pyrrole to imidazole, a symmetricalanhydride protocol, in which pyrrole is activated by formation of thesymmetrical anhydride in the presence of DMAP (Example 5) was developed.The reaction of the activated imidazole acid is extremely rapid, withdilute solutions (<0.1 M) reacting to completion in the standardcoupling time when coupling is imidazole amine, pyrrole amine andaliphatic amines. To avoid the reduced reactivity of the imidazoleamine, while taking advantage of the increased reactivity of theimidazole acid a set of dimeric building blocks were prepared.

The dimeric building blocks were prepared with a series of reactionsanalogous to the preparation of the Boc-imidazole monomer and require noflash chromatography. Scheme 4 (Example 11) illustrates this generalmethod with the synthesis of the Boc-γ-ImCOOH (47) and Boc-PyImCOOH (45)dimers. Both dimers can be prepared in multigram quantities withoutchromatography. The Boc-group is introduced to the imidazole amine witha Boc-protected -OBt activated amino acid. The resulting Boc-aminoacid-imidazole-ethyl ester is isolated by precipitation from water, theethyl group removed by alkaline hydrolysis, and the dimer is collectedby filtration after acidification of the reaction mixture. ##STR12##

Solid Phase Synthesis of Cyclic Polyamides

Cyclic polyamides have also been found to bind to DNA. For example, thecyclic polyamide cyclo-(ImPyPy-γ-PyPyPy-γ-), which took more than a yearto synthesize by solution phase methods, has been shown to bind thepredicted target site 5'-WGWWW-3' (W=A or T) with high affinity andmoderate specificity. (Cho et al. (1995) Proc. Natl. Acad. Sci. USA92:10389). The outlined methods for the synthesis of straight chainpolyamides are readily extendible to the synthesis of cyclic polyamides.Using the solid phase peptide synthesis method of this invention cyclicpolyamides can be prepared in large quantities in a matter of days. Atypical synthetic scheme is outlined in Scheme 5 (see Examples 12 and13).

A key intermediate for the solid phase synthesis of cyclic polyamides isthe Boc-protected allyl ester pyrrole monomer (52), in which theN-methyl group is substituted so as to allow attachment to the resin.The synthesis of this monomer is described in Example 12 (Scheme 19).Briefly, methyl 4-nitropyrrole-2-carboxylate (49) (Fanta (1966) Org.Syn. Coll. 4:844; Morgan and Morrey (1966) Tetrahedron 22:57) is reactedwith benzyl-2-bromoacetate in the presence of potassium iodide andanhydrous potassium carbonate to give the nitro-diester (50) in 85%yield. The nitro group is reduced to the amine and the benzyl estersimultaneously reduced to the acid with Pd/C catalyst and H₂. The amineis immediately protected with boc-anhydride and purified by flashchromatography to give the Boc-protected monoacid (51). The methyl esteris then reacted with allyl alkoxide to give the desired monomer (52).The allyl group is stable to both Boc- or Fmoc-chemistry, but is easilyremoved on the solid support with a soluble palladium catalyst to whichthe benzyl ester resin linkage is stable.

Referring back to Scheme 5, a single equivalent of the Boc-protectedallyl ester pyrrole monomer (52), is attached to the Glycine-PAM-resin,to provide Boc-Py(O-allyl)-G-PAM-resin (53), in high yield. Standardmanual solid phase methods, as described above, are then used toassemble the polyamide, H₂ N-γ-ImPyImPy-γ-ImPyImPy(O-allyl)-G-PAM-resin(54). The allyl group is removed with a soluble palladium catalyst toyield acid (55), which is cleaved from the resin withdimethylaminopropylamine. HPLC purification yields 105 mg of polyamidefrom cleavage of 0.25 mmol resin, a 45% yield. A small sample ofprecursor was then cyclized by treatment with diphenylphosphoryl azide(DPPA) in dilute DMF solution to yield the cyclized polyamide (57),which was purified by HPLC to yield 38% of the cyclic polyamide.Illustrative polyamides prepared by this method are set forth in Table4. ##STR13##

Synthesis of Oligonucleotide-minor Groove Polyamide Conjugates

The methods for the synthesis of minor-groove polyamides are alsoreadily extendable to the synthesis of oligonucleotide-minor groovepolyamide conjugates. A typical synthesis of an oligonucleotideminor-groove polyamide is outlined in Scheme 6 (see Example 13).##STR14## The oligonucleotide portion of the molecule (10 μmol) wasassembled on an automated DNA synthesizer using standard DNA cycles, acommercially available 5'-MMT-C6-amino modifier (MMT=monomethoxytrityl)was attached using an extended 10 minute synthesis cycle. (Connolly andRider (1985) Nuc. Acid. Res. 13:4485; Sproat et al. (1987) Nuc. Acid.Res. 15:4837; Juby et al. (1991) Tet. Lett. 32:879-882). The MMT groupwas removed from the modified oligonucleotide by manual treatment with3% trichloroacetic acid (TCA) in dichloromethane. The controlled poreglass support was then removed from the synthesis cartridge andtransferred to a standard peptide synthesis reaction vessel. Theoligonucleotide (58) was reacted with a 0.2 M solution of Boc-Py-OBt inDMF/0.4M DIEA for 45 minutes. The reaction was determined to be completeby the quantitative ninhydrin test, which showed a distinct blue colorfor the oligonucleotide-polyamide conjugate (59), consistent with a 0.05mmol/gram loading, and a lack of a blue color after 1 hour of reactiontime. The Boc-group was removed with 65% TFA/CH₂ Cl₂ /0.5M PhSH for 20minutes and a second Boc-protected pyrrole coupled to form the aromaticcarboxamide (60). The Boc-group is removed with TFA and the polyamidecapped with N-methylimidazole-2-carboxylic acid. Theoligonucleotide-polyamide conjugate (61) was then simultaneously cleavedfrom the resin and deprotected by treatment with 0.1M NaOH at 55° C. for15 hours and purified by FPLC chromatography.

A single reversed phase purification yields the polyamide conjugate,ImPyPy-CONH(CH₂)₆ -P(O)₄ TTTTTT^(m) C^(m) CTTT-3' (62)(SEQ ID NO:7)(^(m) C=methylcytidine), in high purity. The product obtained ischaracterized by a number of techniques (data not shown). MALDI-time offlight mass spectroscopy shows a single peak corresponding to amolecular mass of 3813.5 (predicted mass of 3814.3), indicating thatfull length product has been isolated. Reverse phase HPLC analysis of 10nmoles of the conjugate, exhibits one major product, absorbing at botholigonucleotide wavelength (260) and polyamide wavelength (340).Enzymatic digestion and subsequent HPLC analysis of a 10 nmole sample ofconjugate is consistent with the proposed composition of theoligonucleotide.

Ultraviolet spectroscopy indicates an additive spectra as might beexpected for a conjugate of 2-imidazole Netropsin and an 11-merthymidine rich oligonucleotide. From the extinction coefficient of thebases, 8,800 for thymidine (T) at 260 nm and 5,700 for methylcytidine(MeC) at 260 nm and the reported extinction coefficients for 2-ImN of19,000 (255 nm) and 26,000 (302 nm), it is possible to predict the ratioof the extinction coefficients at 260 nm. (Colocci et al. (1993) J. Am.Chem. Soc. 115:4468-4473). Assuming a contribution from the oligo of90,600 and from the polyamide of 19,000, a ratio of 4.2 is expected anda ratio of 3.7 is observed.

Finally, a 14 mg sample of the polyamide conjugate was dissolved in 700μl of deuterium oxide and analyzed by ¹ H NMR spectroscopy at 300 MHz.Although most of the spectrum is complex, the aromatic region is readilyinterpreted. The protons expected in the aromatic region correspond tothe polyamide ring protons, and the C₆ ring protons of thymidine and5-methylcytidine. The observed spectrum is consistent with the predictedsequence ImPyPy-CONH(CH₂)₆ -P(O)₄ TTTTTT^(m) C^(m) CTTT-3', with the 2protons observed at 7.7 corresponding to the cytidine, 9 protons at 7.6corresponding to the thymidine contribution, 2 protons at 7.3corresponding to the imidazole ring, four pyrrole doublets at 7.2, 7.1,6.9, and 6.7 corresponding to four protons, and 11 anomeric protons at6.2. The purity of the sample, as determined by NMR, is >98%. Theability to rapidly obtain NMR data (30 minutes of acquisition) on amolecule of this size (3 kD) warrants a synthesis scale such as the onechosen here. Table 5 sets forth illustrative oligonucleotide-polyamideconjugates synthesized by the method of this invention.

In another embodiment (Scheme 7), the oligonucleotide, prepared usingstandard phosphoramidite chemistry, is capped with a 2',5'-dideoxy-5'-aminothymidine (Smith et al. (1985) Nucleic Acids Research13:2399), prior to attachment of the polyamide (68).

The free amino group of the oligonucleotide is then reacted with the bisNHS ester of glutaric acid (DMF/DIEA) for 2 hours at room temperature toform activated acid (69). Excess NHS ester is removed by washing with alarge excess of DMF. The activated acid is then treated with anequivalent of a polyamide containing a free amine prepared by the methodof this invention. The coupling reaction (DMF/DIEA) is allowed toproceed for 12 hours, and any unreacted polyamide is removed by washingthe resin. The oligonucleotide (70) is deprotected and simultaneouslycleaved from the resin with a solution of 0.1 M NaOH at 55° C. for 12hours. The polyamide-oligonucleotide conjugate (71) is then purified bya single reverse phase chromatography step (C18, TEAA, pH 7), to give a10% yield. A list of illustrative polyamide-oligonucleotide conjugateswhich have been prepared by the method of this invention is set forth inFIG. 18 and Table 5. FIG. 19 depicts a ribbon graphic illustrating howthe conjugate Dp-G-PyPyPy-G-PyPyIm-linker-TTTTTT^(m) C^(m) CTTT-3' mightbind to the double helical DNA. ##STR15##

Preparation of Modified Derivatives

In yet another embodiment of this invention, modifiedpolyamide-oligonucleotide conjugates having terminal 1°, 2° and 3° aminogroups are prepared. Modifications include, but are not limited to theformation of the dimethylaminopropylamine (Dp), the γ-aminobutyric acid(γ), the ethylenediamine (ED), the 3, 3'-diamino-N-methylpropylamine(Ta) or the ethylenediaminetetraacetic acid (EDTA) derivatives.

Scheme 7 illustrates this method by the formation of the γ-aminobutyricderivative (75) which is then further modified by formation of the EDTAderivative (76). ##STR16## Referring to Scheme 7, the conjugateBoc-ImPyPy-CONH(CH₂)₁₂ -P(O)₄ -TTTTTT^(m) C^(m) CTTT-CPG (72) issynthesized on solid support as described above. The Boc-group isremoved with TFA under standard conditions and the product is reactedwith the HOBt ester of Boc-γ (generated in situ). The product,Boc-γ-ImPyPy-CONH(CH₂)₁₂ -P(O)₄ -TTTTTT^(m) C^(m) CTTT-CPG (73) issimultaneously cleaved from the resin and the bases deprotected bytreatment with 0.1M NaOH at 55° C. for 15 hours. The mixture is thenpurified by FPLC to give Boc-γ-ImPyPy-CONH(CH₂)₁₂ -P(O)₄ -TTTTTT^(m)C^(m) CTTT-3' (SEQ ID NO:12) (74) which is characterized by HPLC,enzymatic degradation and mass spectroscopy. The Boc-group is thenremoved by treatment with TFA under standard conditions and the mixturepurified by FPLC to give H₂ N-γ-ImPyPy-CONH(CH₂)₁₂ -P(O)₄ -TTTTTT^(m)C^(m) CTTT-3' (75) (SEQ ID NO:10) which is characterized by massspectroscopy. The oligonucleotide-polyamide conjugate is then reactedwith the monoanhydride of EDTA in pH 9.5 carbonate buffer to yield theEDTA derivative (76) (SEQ ID NO:11).

Synthesis of Protein-minor Groove Polyamide Conjugates

In another embodiment of this invention the method of preparingpolyamides is extended to the preparation of polyamides that areattached to a protein (referred to herein as a protein-polyamideconjugate). This method is illustrated by the replacement of the DNAbinding domain of the major groove DNA binding protein GCN-4 (Oakley andDervan (1989) Science 248:847) with the polyamide, NH₂-β-β-ImPyPy-γ-PyPyPy-γ-γ-, prepared as described above. A ribbon modelof GCN-4 is depicted in FIG. 20. As illustrated in FIG. 20 the first 50residues comprise an α-helix which has a DNA binding domain and a coiledcoil dimerization region. The coiled-coil region--Lys Gln Leu Glu AspLys Val Glu Glu Leu Leu Ser Lys Asn Tyr His Leu Glu Asn Glu Val Ala ArgLeu Lys Lys Leu Val Gly Glu Arg-CO₂ NH₂ (SEQ ID NO:24)--is preparedusing standard NMP/HOBt methods for an ABI 430A peptide synthesizer. Thepolyamide domain is then appended in a stepwise fashion using thesynthetic methods described above. The conjugate H₂N-β-β-ImPyPy-γ-γ-protein (SEQ ID NO:25) is then cleaved from the resinwith anhydrous HF, and purified by reversed phase HPLC chromatography,to provide 1.1 mg of conjugate from cleavage of 100 mg of resin. Theconjugate has been characterized by analytic HPLC and mass spectroscopy.FIG. 21 illustrates the DNA binding domain of the GCN-4-polyamideconjugate.

The ability to easily prepare polyamides with an appended peptide moietyis useful, since peptide leader sequences often provide a means fordelivering molecules into cells. (Soukchareun (1995) J. Bioconj Chem.6,1:43-53)

Automated Synthesis of Polyamides

The methods of this invention for the syntheses of polyamides aresuitable for automation. A peptide synthesizer 110, shown in FIG. 22,was modified to prepare polyamides containing imidazole and pyrrolecarboxamides. The peptide synthesizer 110 has three chemistry centerswhere the reactions occur: an activator center 112, a concentratorcenter 114 and a reaction center 118. The activator center (firstchemistry center) 112 is not used in preparing the polyamides. Theconcentrator center (second chemistry center) 114 is coupled to theactivator center 112 by a tube 116. The reaction center (third chemistrycenter) 118 is coupled to the concentrator center 114 by a tube 120. Afirst valve 122 controls the flow of the contents from the activatorcenter 112 to the concentrator center 114. A second valve 123 controlsthe flow of the contents from the concentrator center 114 to thereaction center 118. Both valves 122, 123 are coupled to a controller124, that provides signals that control both valves 122, 123.

Another valve 125, operated by the controller 124, connects the reactioncenter 118 to a drain 126. A shaker 127, operated by the controller 124,shakes the reaction center 118. The three chemistry centers 112, 114 and118 are coupled to a plurality of reagents by a valve matrix 128. Thereagents are contained in a plurality of bottles 130 in one of tenreagent positions. The valve matrix allows any of the reagents inreagent positions 1-10 to flow into any of the three chemistry center112, 114, 118. The valve matrix has programmable valves that arecontrolled by the controller 124. The pressurized air source 32 allowsaeration of each of the three chemistry center 112, 114 and 118.

A plurality of cartridges 134 typically containspredissolved/preactivated monomer units. A programmable needle 36transfers the contents of the cartridges 134 to one of the plurality ofchemistry center 112, 114 and 118. The programmable needle 136 isdirected by the controller 124.

The controller 124 is connected to a floppy disk drive 138. The floppydisk drive 138 accepts a floppy disk 140 having a storage medium encodedwith a computer program to direct the operation of the peptidesynthesizer 110. FIG. 23 is a flow chart of a computer program used toproduce polyamides containing imidazole and pyrrole carboxamides in thepeptide synthesizer 110. The process begins at block 200. First,preactivated monomer is dissolved in DMF and transferred from one of theplurality of cartridges 134 to the concentrator 114, at block 202,bypassing the activator center 112. DIEA, from one of the plurality ofreagent bottles 130, is transferred to the concentrator 114, at block204. Simultaneously, a resin in the reaction center 118 is treated withTFA and PhSH, at block 206. The TFA and PhSH are contained in thereagent bottles 30. The resin is used to support the growing polyamide.Next, the TFA is drained for the reaction center 118, at block 208.Dichloromethane and TFA washes are performed before and after TFA/PhSHtreatment. The dichloromethane and PhSH are contained in the pluralityof reagent bottles 130. Following the dichloromethane/TFA wash, thecontents of the concentrator 14 are transferred to the reaction center118, at block 210. At block 212, a shaker starts shaking the reactioncenter 118 and a timer in the controller is started. At t=1800s DMSOfrom one of the plurality of reagent bottles is added to the reactioncenter and DIEA from one of the plurality of reagent bottles is added tothe reaction center, at block 214. The reaction center 118 is thendrained, at block 216. The reagent Ac₂ O is added to the reaction centerat block 218. The reaction center 118 is then drained again at block220. At block 222, it is determined if a timer is greater than or equalto two hours. The process waits at block 222 until the timer equals orexceeds two hours. Then the shaking is stopped at block 224 and theprocess ends at block 226.

The machine assisted protocols are highly efficient as demonstrated bythe synthesis of the 8 residue polyamide ImPyPy-γ-PyPyPy-β-Dp, with thecrude reaction product containing >70% of the desired polyamide asdetermined by HPLC analysis. It is possible, however, to modify othercommercially available peptide synthesizers and organic synthesismachines to accommodate the automated chemistry performed to synthesizethe polyamides of this invention.

The versatility of the machine assisted protocols is demonstrated by thesynthesis of polyamides H₂N-β-PyPyPy-γ-ImImPy-β-β-β-β-PyPyPy-γ-ImImPy-β-Dp and H₂N-β-PyPyPy-γ-ImImPyPy-β-β-PyPyPyPy-γ-ImImPy-.beta.-Dp both of which werecharacterized by HPLC, ¹ H NMR, and MALDI-TOF mass spectroscopy. FIG. 24depicts a ball and stick model of the projected binding mode ofpolyamide (#) with the target sequence 5'-TGGTTAGTACCT-3' (SEQ ID NO:5)on PXLO-wt. The binding affinity was determined to be approximately1×10⁹ M⁻¹. Introduction of a single base pair mismatch in the bindingsite lowers affinity by apprxomately a factor of 10-fold.

EXAMPLES

Materials

Di-t-butyldicarbonate (Boc-anhydride),Boc-G-(-4-carboxamidomethyl)-benzyl-ester-copoly(styrene-divinylbenzene)resin [Boc-G-PAM-resin] (0.2 mmol/gram), dicyclohexylcarbodiimide (DCC),hydroxybenzotriazole (HOBt) and aminomethylated polystyrene resin (0.7mmol/gram) were purchased from Peptides International (Louisville, Ky.).N,N-diisopropylethylamine (DIEA), N,N-dimethylformamide (DMF), aceticanhydride, N-methylpyrrolidone (NMP), 0.0002M potassiumcyanide/pyridine, dimethylsulfoxide (DMSO) and DMSO/NMP were purchasedfrom Applied Biosystems. Boc-Glycine (Boc-G) was purchased fromPeninsula, Boc-γ-aminobutyric acid (Boc-γ) from NOVA Biochem,dichloromethane from EM, thiophenol (PhSH) and picric acid from Aldrich,trifluoroacetic acid (TFA) from Halocarbon, phenol from Fisher, andninhydrin from Pierce. Unless stated otherwise reagent-grade chemicalswere used. Additionally, all reagents were used without furtherpurification. Quik-Sep polypropylene disposable filters were purchasedfrom Isolab Inc. and were used for filtration of dicyclohexylurea (DCU)for washing the resin for the ninhydrin and picric acid tests. A shakerfor manual solid phase synthesis was obtained from Milligen. Screw-capglass peptide synthesis reaction vessels (5 ml) with a #2 scinteredglass frit were made at the California Institute of Technology glassshop as described by Kent. (Kent (1988) Ann. Rev. Biochem. 57:957).

DNA Reagents and Materials

Sterilized 0.1% DEPC-treated water (Sambrock et al. (1989) MolecularCloning, 2nd ed.; Cold Spring Harbor, N.Y.) was either prepared orpurchased from Gibco. Polyacrylamide gel electrophoresis was performedin a 1×TBE buffer. Autoradiography was carried out using AmershamHyperfilm MP or Kodak X-Omat film. Gels were analyzed by storagephosphor technology. (Miyahara et al. (1986) Nuc. Inst. Meth. Phys. Res.A246:572-578; Johnston et al. (1990) Electrophoresis 11:355-360).

NMR were recorded on a GE 300 instrument operating at 300 MHz (¹ H) and75 MHz (¹³ C). Chemical shifts are reported in ppm relative to thesolvent residual signal. UV spectra were measured on a Hewlett-PackardModel 8452A diode array spectrophotometer. Matrix-assisted, laserdesorption/ionization time of flight mass spectrometry was carried outat the Protein and Peptide Microanalytical Facility at the CaliforniaInstitute of Technology. HPLC analysis was performed either on a HP1090M analytical HPLC or a Beckman Gold system using a RAINEN C₁₈,Microsorb MV, 5 μm, 300×4.6 mm reversed phase column in 0.1% (wt/v) TFAwith acetonitrile as eluent and a flow rate of 1.0 ml/min, gradientelution 1.25% acetonitrile/min. Preparatory HPLC was carried out on aBeckman HPLC using a Waters DeltaPak 25×100 mm, 100 μm C₁₈ columnequipped with a guard, 0.1% (wt/v) TFA, 0.25% acetonitrile/min. 18MΩwater was obtained from a Millipore MilliQ water purification system,all buffers were 0.2 μm filtered. Flash column chromatography wascarried out using Silica Gel 60 (230-400 mesh, Merck). Thin layerchromatography (TLC) was performed on Silica Gel 60 F₂₅₄ precoatedplates (Merck).

Example 1 Preparation of the Pyrrole and Imidazole Monomers Preparationof Boc-Py-OBt (9) and Boc-Im-OBt (13)

The Boc-Py-OBt (9) and Boc-Im-OBt (13) monomers were synthesizedstarting from the known nitro-esters 6 and 10 (prepared as described inSchemes 10 and 11 (Bailey et al. Org. Syn.101-102; Corwin andQuattlebaum (1936) J. Am. Chem. Soc. 58:1081-1085; Grehn (1978) Chim.Scripta 13:67-77; Morrey and Morrey (1966) Tetrahedron 22:57-62;Krowicki and Lown (1987) J. Org. Chem. 52:3493-3501) as outlined inScheme 8 below. Reduction of the nitro group gave amines 7 and 11, in91% and 81% yield respectively. The amines were Boc-protected withBoc-anhydride (pyrrole amine 7 in aqueous carbonate/dioxane andimidazole amine 11 in DMF/DIEA) and the ester groups were hydrolyzedwith aqueous sodium hydroxide to yield the Boc-protected acids 8 and 12in 93% and 88% yields, respectively. The acids were then activated athigh concentration (>0.2 M acid in DMF) with 1 equivalent of DCC andHOBt and the -OBt esters precipitated from water to give compounds 9 and13. Overall yields starting from the nitro/methyl esters arereproducibly greater than 60% for both the pyrrole and imidazole -OBtesters, with simple purification requiring no column chromatography. TheBoc-imidazole acid has been reported to decarboxylate even at reducedtemperature. The Boc-Im-OBt ester 13, however, has been found to bestable at room temperature, with HOBt effectively acting as a protectinggroup for the unstable imidazole carboxylic acid. ##STR17##

Preparation of Methyl 4-amino-1-methyl-pyrrole-2-carboxylatehydrochloride (7)

Recrystallized methyl 1-methyl-4-nitropyrrole-2-carboxylate 6 (40 g,0.22 mol) was dissolved in 900 ml ethyl acetate. A slurry of 10 g of 10%Pd/C in 100 ml ethyl acetate was added and the mixture stirred under aslight positive pressure of hydrogen (about 1.1 ATM) for 48 hours. ThePd/C was removed by filtration through Celite, washed with 50 ml ethylacetate, and the volume of the mixture was reduced to about 200 ml. 700ml of ethyl ether was added and HCl gas gently bubbled through themixture while maintaining a temperature below 20° C. The precipitatedamine hydrochloride was then collected after storage at -20° C. for 40hours to yield (38 g, 91%) of a very white powder. TLC (ethyl acetate)Rf amine (0.6), Rf salt (0.0); ¹ H NMR (DMSO-d₆) δ 10.23 (br s, 3H),7.24 (d, 1H, J=1.9 Hz), 6.79 (d, 1H, J=2.0 Hz), 3.83 (s, 3H), 3.72 (s,3H); ¹³ C NMR (DMSO-d₆) δ 160.8, 124.3, 121.2, 113.4, 112.0, 51.8, 37.1.

Preparation of4-[[(tert-Butyloxy)carbonyl]-amino]-1-methylpyrrole-2-carboxylic acid(8)

The hydrochloride salt of the pyrrole amine 7 (24 g, 146 mmol) wasdissolved in 300 ml of 10% aqueous sodium carbonate anddi-t-butyldicarbonate (40 g, 174 mmol) slurried in 75 ml of dioxane wasadded over a period of ten minutes at room temperature. The reaction wasallowed to proceed at room temperature for two hours and then cooled to5° C. for 2 hours. The resulting white precipitate was collected byvacuum filtration. The crude product was dissolved in 350 ml MeOH and350 ml of 1M NaOH was added and the solution was heated at 60° C. for 6hours. The reaction was then cooled to room temperature, washed withethyl ether (2×500 ml). The pH of the aqueous layer was reduced toapproximately 3 with aqueous citric acid and was extracted with ethylacetate (4×500 ml). The combined ethyl acetate extracts were dried withsodium sulfate and concentrated in vacuo to give a tan foam. The foamwas dissolved in 100 ml of dichloromethane and 400 ml petroleum etherwas added and the resulting slurry was concentrated in vacuo. This stepwas repeated three times to give (31 g, 93% yield) of a fine whitepowder. TLC (7:2 benzene/ethyl acetate v/v) Rf (ester) 0.8, Rf (acid)0.1. (ethyl acetate), Rf (acid) 0.6; ¹ H NMR (DMSO-d₆) δ 12.10 (s, 1H),9.05 (s, 1H), 7.02 (s, 1H), 6.55 (s, 1H), 3.75 (s, 3H), 1.41 (s, 9H); ¹³C NMR (DMSO-d₆) δ 162.4, 153.2, 123.3, 120.1, 119.2, 107.9, 78.9, 36.6,28.7.

Preparation of 1,2,3-Benzotriazol-1-yl4[[(tert-Butyloxy)carbonyl]-amino]-1-methylimidazole-2-carboxylate (9)

The Boc-pyrrole-acid 8 (31 g, 129 mmol) was dissolved in 500 ml DMF andhydroxybenzotriazole (17.4 g, 129 mmol) was added followed by DCC (34 g,129 mmol). The reaction was allowed to stir for 24 hours and thenfiltered dropwise into a well stirred solution of 5 liters of water (0°C.). The precipitate was allowed to sit for 15 minutes at 0° C. and thencollected by filtration. The wet cake was dissolved in 500 ml ofdichloromethane, washed with 200 ml brine, and added slowly to a stirredsolution of petroleum ether at -20° C. After 4 hours at -20° C. theprecipitate was collected by vacuum filtration and dried in vacuo togive (39 g, 85% yield) of a finely divided white powder. (A yellowishimpurity may be observed, which can be removed by flash chromatography(acetone:dichloromethane), followed by precipitation in petroleumether). TLC (7:2 benzene/ethyl acetate v/v) Rf 0.6; ¹ H NMR (DMSO-d₆) δ9.43 (s, 1H), 8.12 (d, 1H, J=8.4Hz), 7.80 (d, 1H, J=8.2 Hz), 7.64 (t,1H, J=7.0 Hz), 7.51 (m, 2H), 7.18 (s, 1H), 3.83 (s, 3H), 1.45 (s, 9H);¹³ C NMR (DMSO-d₆) δ 156.5, 153.3, 143.2, 129.6, 129.2, 125.7, 125.2,124.6, 120.3, 112.8, 110.3, 109.8, 79.5, 36.8, 28.6.

Preparation of Ethyl 4-amino-1-methylimidazole-2-carboxylatehydrochloride (11)

Nitro imidazole ethyl ester 10 (10 g, 50 mmol) was dissolved in 500 mlof 1:1 ethanol/ethyl acetate, 1 g 10% Pd/C slurried in 50 ml ethylacetate was added and the mixture was stirred under a slight positivepressure of hydrogen (approximately 1.1 atm) for 48 hours. The reactionmixture was filtered, concentrated in vacuo and dissolved in 600 mlether. HCl gas was bubbled through the ether solution at 0° C. to give awhite precipitate. The solution was cooled at -20° C. for 4 hours andthe precipitate was collected by vacuum filtration and dried in vacuo togive (8.1 g, 81% yield) of 11 as a fine white powder. ¹ H NMR (DMSO-d₆)δ 10.11 (br s, 3H), 7.43 (s, 1H), 4.28 (q, 2H, J=7.1 Hz), 3.92 (s, 1H),1.28 (t, 3H, J=7.1 Hz); ¹³ C NMR (DMSO-d₆) δ 157.6, 132.6, 117.4, 117.3,61.8, 36.6, 14.5.

Preparation of4-[[(tert-Butyloxy)carbonyl]-amino]-1-methylimidazole-2-carboxylic acid(12)

The imidazole amine 11 (3 g, 14.5 mmol) was dissolved in 20 ml DMF anddiisopropylethylamine (4.5 ml, 25 mmol) was added followed bydi-t-butyldicarbonate (6 g, 27 mmol). The mixture was shaken at 40° C.for 18 hours, allowed to return to room temperature and then partitionedbetween 500 ml of brine and 500 ml of ethyl ether. The ether layer wasextracted with (2×200 ml each) 10% citric acid, brine, saturated sodiumbicarbonate and brine. The ether layer was dried over sodium sulfate andconcentrated in vacuo to yield the Boc-ester. The crude Boc-ester wasdissolved in 40 ml of MeOH and 40 ml of 1 M KOH was added. The reactionmixture was shaken at 40° C. for 4 hours, cooled to room temperature andpartitioned between 200 ml of water and 300 ml ethyl ether. The aqueouslayer was washed with 300 ml ethyl ether, the ether washes werediscarded, and the pH of the aqueous layer was brought down toapproximately 3 with 10% aqueous sodium bisulfate. The aqueous layer wasextracted (10×150 ml) with ethyl acetate and the organic layers werecombined, dried over sodium sulfate and concentrated in vacuo to yieldpure 12 as a white chalky powder (3.1 g, 88% yield). ¹ H NMR (DMSO-d₆) δ9.61 (s, 1H), 7.23 (s, 1H), 3.85 (s, 3H), 1.41 (s, 9H).

Preparation of 1,2,3-Benzotriazol-1-yl4[[(tert-butyloxy)carbonyl]-amino]-1-methylpyrrole-2-carboxylate (13)

The Boc-imidazole-acid 12 (2 g, 8.3 mmol) was in dissolved in 10 ml ofDMF and 1-hydroxybenzotriazole was added (1.2 g, 9 mmol) followed by DCC(2.4 g, 9 mmol). After 6 hours the precipitate was removed by filtrationand washed with 4 ml of DMF. The DMF solution was added dropwise to 250ml of well stirred ice water and the resulting precipitate was collectedby vacuum filtration. The filter cake was ground and dried in vacuo overP₂ O₅ to give (2.7 g, 89%) of 13 as a pale yellow power contaminatedwith a small amount of DCU (2.7 g, 89%). ¹ H NMR (DMSO-d₆) δ 9.62 (s,1H), 7.96 (s, 1H), 7.68 (d, 1H), 7.52 (d, 1H), 7.38 (d, 1H), 7.23 (s,1H), 3.85 (s, 3H), 1.33 (s, 9H).

Preparation of Methyl 1-methyl-4-nitropyrrole-2-carboxylate (6) andEthyl 1-methyl-4-nitroimidazole-2-carboxylate (10)

Nitroesters 6 and 10 were synthesized from the inexpensiveN-methylpyrrole and N-methylimidazole as outlined in Schemes 10 and 11,respectively. Each of these compounds can be prepared economically on alarge scale. Methyl 1-methyl-4-nitropyrrole-2-carboxylate (6) wasprepared using a modification of the reported synthesis ofpyrrole-2-trichloroketone. (Bailey et al. (1971) Org. Syn. 51:101).Briefly, reaction of the inexpensive N-methylpyrrole (14) withtrichloroacetylchloride followed by nitration with nitric acid gavenitropyrrole trichloroketone (15), which was treated with sodiummethoxide to yield nitropyrrole (6). ##STR18##

Preparation of 4-nitro-2-trichloroacetyl-1-methylpyrrole (15)

To a well stirred solution of trichloroacetylchloride (1 kg, 5.5 mole)in 1.5 liters of ethyl ether was added dropwise over a period of 3 hoursa solution of N-methylpyrrole (14) (0.45 kg, 5.5 mole) in 1.5 liters ofanhydrous ethyl ether. The reaction was allowed to stir for anadditional 3 hours and quenched by the dropwise addition of a solutionof 400 g of potassium carbonate in 1.5 liters of water. The layers wereseparated and the ether layer was concentrated in vacuo to provide2-trichloroacetyl pyrrole (1.2 kg, 5.1 mole) as a yellow crystallinesolid, which can be purified by recrystallization from benzene, but issufficiently pure to be used without further purification. To a cooled(-40° C.) solution of 2-trichloroacetyl pyrrole (1.2 kg, 5.1 mole) inacetic anhydride (6 liters) flask equipped with a mechanical stirrer wasadded 440 ml of fuming nitric acid over a period of 1 hour whilemaintaining a temperature of -40° C. The reaction was carefully allowedto warm to room temperature and stirred an additional 4 hours afterwhich the mixture was cooled to -30° C. and isopropyl alcohol (6 liters)was added. The solution was stirred at -20° C. for 30 minutes duringwhich time a white precipitate formed. The solution was allowed to standfor 15 minutes and the resulting precipitate collected by vacuumfiltration to provide (15) (0.8 kg, 54% yield). TLC (7:2 benzene/ethylacetate) Rf 0.7; ¹ H NMR (DMSO-d₆) δ 8.55 (d, 1 H, J=1.7 Hz), 7.77 (d, 1H, J=1.7 Hz), 3.98 (s, 3 H); ¹³ C NMR (DMSO-d₆) δ 173.3, 134.7, 133.2,121.1, 116.9, 95.0, 51.5; IR (KBr) 1694, 1516, 1423, 1314, 1183, 1113,998, 750; FABMS m/e 269.936 (M+H 269.937 calc. for C₇ H₅ N₂ O₃ Cl₃).

Preparation of Methyl 1-methyl-4-nitropyrrole-2-carboxylate (6)

To a solution of (15) (800 g, 2.9 mol) in 2.5 liters of methanol in aflask equipped with a mechanical stirrer was added dropwise a solutionof NaH (60% dispersion in oil) (10 g, 0.25 mole) in 500 ml of methanol.The reaction was allowed to stir for 2 hours at room temperature, andquenched by the addition of concentrated sulfuric acid (25 ml). Thereaction was then heated to reflux, until a clear light yellow solutionformed. The reaction was slowly cooled to room temperature as (6)crystallizes as white needles, which are collected by vacuum filtrationand dried in vacuo to provide 450 g (47% yield). TLC (ethyl acetate) Rf0.8; ¹ H NMR (DMSO-d₆) δ 8.22 (d, 1 H, J=1.7 Hz), 7.22 (d, 1 H, J=1.6Hz), 3.88 (s, 3 H), 3.75; ¹³ C NMR (DMSO-d₆) δ 37.8, 52.2, 112.0, 123.0,129.9, 134.6, 160.3; IR(KBr) 3148, 1718, 1541, 1425, 1317, 1226, 1195,1116, 753; FABMS m/e 183.048 (M+H 184.048 calc. for C₇ H₈ N₂ O₄).

Ethyl 1-methyl-4-nitroimidazole-2-carboxylate (10) was prepared bytreatment of N-methylimidazole (16) with ethylchloroformate andtriethylamine followed by nitration with nitric acid. ##STR19##

Preparation of Ethyl 1-methyl-imidazole-2-carboxylate (17)

N-methylimidazole (16) (320 g, 3.9 mol) was combined with 2 liters ofacetonitrile and 1 liter of triethylamine in a flask equipped with amechanical stirrer and the solution was cooled to -20° C.Ethylcloroformate (1000 g, 9.2 mol) was added with stirring, keeping thetemperature between -20° C. and -25° C. The reaction was allowed toslowly warm to room temperature and stir for 36 hours. Precipitatedtriethylamine hydrochloride was removed by filtration and the solutionwas concentrated in vacuo at 65° C. The resulting oil was purified bydistillation under reduced pressure (2 torr, 102° C.) to provide (17) asa white solid (360 g, 82% yield). TLC (7:2 benzene/ethyl acetate) Rf0.2; ¹ H NMR (DMSO-d₆) δ 7.44 (d, 1 H, J=2.8 Hz), 7.04 (d, 1 H, J=2.8Hz), 4.26 (q, 2 H, J=3.5 Hz) 3.91 (s, 3 H), 1.26 (t, 3 H, J=3.5 Hz); ¹³C NMR (DMSO-d₆) δ 159.3, 129.1, 127.7, 61.0, 36.0, 14.5; IR(KBr) 3403,3111, 2983, 1713, 1480, 1422, 1262, 1134, 1052, 922, 782, 666; FABMS m/e155.083 (M+H 155.083 calc. for C₇ H₁₁ N₂ O₂).

Preparation of Ethyl 4-nitro-1-methylimidazole-2-carboxylate (10)

Compound (17) was carefully dissolved in 1000 ml of concentratedsulfuric acid cooled to 0° C. 90% nitric acid (1 liter) was slowly addedmaintaining a temperature of 0° C. The reaction was then refluxed withan efficient condenser (-20° C.) in a well ventilated hood for 50minutes. The reaction was cooled with an ice bath, and quenched bypouring onto 10 liters of ice. The resulting blue solution was thenextracted with 20 liters of dichloromethane and the combined extractswere dried and concentrated in vacuo to yield a tan solid which wasrecrystallized from 22 liters of 21:1 carbon tetrachloride/ethanol. Theresulting white crystals were collected by vacuum filtration to providepure (10). (103 g, 22% yield). TLC (7:2 benzene/ethyl acetate) Rf 0.5, ¹H NMR (DMSO-d₆) δ 8.61 (s, 1 H), 4.33 (1, 2 H, J=6.4 Hz), 3.97 (s, 3 H),1.29 (t, 3 H, J=6.0 Hz); ¹³ C NMR (DMSO-d₆) δ 158.2, 145.4, 135.3,127.4, 62.2, 37.3, 14.5; IR(KBr) 3139, 1719, 1541, 1508, 1498, 1381,1310, 1260, 1147, 1122, 995, 860, 827, 656; FABMS m/e 200.066 (M+H200.067 calc. for C₇ H₁₀ N₃ O₄).

Synthesis of Fmoc-Py-OBt (21a) and Fmoc-Im-OBt (21b)

The Fmoc-monomers were synthesized from the Boc-monomers as set forth inScheme 12. Briefly, the Boc-protected monomer is converted to the cesiumsalt followed by treatment with benzyl bromide to yield the benzylesters (18). The Boc-protecting group is then removed withtrifluoroacetic acid in the presence of thiophenol and the productprecipitated by the addition of HCl saturated ethyl ether. The aminehydrochloride is then treated with Fmoc-chloroformate in potassiumcarbonate, and the benzyl ester removed by hydrogenation to provide theFmoc-protected monomers (21a and 21b). This method is illustrated usingthe synthesis of the Fmoc-protected pyrrole (21a) as an example.##STR20##

Preparation of Benzyl4-[[tert-butyloxy)carbonyl]amino]-1-methylpyrrole-2-carboxylate (18a, b)

To a solution of Boc-acid (8) (5 g) in 100 ml of 66% ethanol was addedcesium carbonate (3.3 g in 25 ml of water). The solution was stirred for20 minutes, filtered through glass wool, and concentrated in vacuo toyield the cesium salt as a solid. The solid was dissolved in 75 ml ofethanol and concentrated to dryness three times. The cesium salt wasthen dissolved in 500 ml of DMF and 2.6 ml of benzyl bromide wasimmediately added dropwise. The resulting solution was stirred at 40° C.for 10 hours. After 10 hours the solution was poured into 300 ml of icewater and allowed to stand at 4° C. for 1 hour. The resultingprecipitate was collected by vacuum filtration to yield 6.59 (93%) ofthe Boc protected benzyl ester (18a). ¹ H NMR (DMSO-d₆) δ 9.1 (s, 1 H),7.4 (m, 5 H), 7.1 (d, 1 H), 6.8 (d, 1 H), 5.2 (s, 2 H), 3.8 (s, 3 H),1.4 (2, 9 H).

Preparation of Benzyl 4-amino-1-methylpyrrole-2-carboxylate (19a)

To a solution of the Boc-benzyl ester(18a) (5 g) in 20 ml ofdichloromethane was added 20 ml of 65% TFA/CH₂ Cl₂ /0.5 M PhSH. Thereaction was allowed to stir for 1 hour, and then partitioned between100 ml of 1M LiOH and 100 ml of ethyl ether. The layers were separatedand the aqueous layer was extracted with ethyl ether (5×20 ml). HCl (g)was bubbled through the combined organics and the product collected byvacuum filtration to yield 3.2 g (76%) of benzylamine (19a). ¹ H NMR(DMSO-d₆) δ 10.1 (br s, 3 H), 7.4 (m, 5H), 7.2 (d, 1 H), 6.8 (d, 1 H),5.2 (s, 2 H), 3.8 (s, 3 H).

Preparation of4-[[(9-fluorenylmethyl)carbonyl]amino]-1-methylpyrrole-2-carboxylate(20a)

To a solution of the benzylamine (19a) (1 gram) in dichloromethanecooled at 0° C. was added DIEA (1.4 ml) and9-fluorenylmethylchloroformate (973 mg). The reaction was allowed tostir for 30 minutes. The reaction was worked up using standard methodsto yield 1.8 g (88%) of benzylester (21a). ¹ H NMR (DMSO-d₆) δ 9.5 (s, 1H), 7.9 (d, 2 H), 7.7 (d, 2 H), 7.3 (m, 9 H), 7.1 (s, 1 H), 6.7 (s, 1H), 5.2 (s, 2 H), 4.4 (d, 2 H), 4.2 (t, 1 H), 3.8 (s, 3 H).

Preparation of4-[[(9-fluorenylmethyl)carbonyl]amino]-1-methylpyrrole-2-carboxylic acid(21a)

To a solution of the benzylester (20a) (900 mg) dissolved in THF (10 ml)was added 10% Pd/C (100 mg). The solution was hydrogenated (1 atm) for19 hours and worked up using standard methods to yield 580 mg (80%) ofcompound (21a). ¹ H NMR (DMSO-d₆) δ 9.4 (s, 1 H), 8.0 (m, 2 H), 7.8 (m,2 H), 7.3 (m, 4 H), 7.1 (s, 1 H), 6.8 (s, 1 H), 4.6 (m, 2 H), 4.3 (m, 1H), 3.8 (s, 3 H).

Preparation of N-substituted Monomers Preparation ofN-2-methyl-butyl-4-[[(tert-butyloxy)carbonyl]amino]-2-carboxylic acid(24)

N-2-methyl-propyl-4-[[(tert-butyloxy)carbonyl]amino]-2-carboxylic acid24 was synthesized as outlined in Scheme 13. Briefly, methyl4-nitropyrrole-2-carboxylate 22, prepared as described below, wasalkylated by refluxing with the appropriate alkyl halide in acetone inthe presence of potassium carbonate. The ester was then hydrogenated andhydrolyzed to provide the modified monomer 24 which is ready for use insolid phase synthesis. ##STR21##

Preparation of Methyl N-2-methyl-butyl-4-nitropyrrole-2-carboxylate (23)

Methyl 3-nitropyrrole-2-carboxylate (22) (2.7 g, 15.9 mmol), potassiumcarbonate (6.5 g), and iodo-2-methylbutane (5.2 ml) were dissolved in100 ml of acetone and refluxed for 10 hours. The reaction mixture wasthen cooled to room temperature, concentrated in vacuo, partitionedbetween 200 ml of dichloromethane and 200 ml of water and extracted withdichloromethane (2×200 ml). The combined organic layers were dried(sodium sulfate) and concentrated in vacuo. The resulting yellow oil waspurified by flash chromatography to provide the substituted nitropyrrole 23 (2.5 g, 70% yield). ¹ H NMR (DMSO-d₆) δ 8.30 (d, 1 H, J=2.0Hz), 7.33 (d, 1 H, J=1.9 Hz), 4.15 (d, 2 H, J=7.4 Hz), 3.77 (s, 3 H),2.03 (m, 1 H, J=3.1 Hz), 0.80 (d, 6 H, J=6.7 Hz); ¹³ C NMR (DMSO-d₆) δ160.30, 134.79, 129.80, 122.40, 112.76, 105.00, 56.63, 52.41, 29.72,19.73; FABMS, 226.096 calcd, 226.095 found.

Preparation ofN-2-methylbutyl-4-[[(tert-butyloxy)carbonyl]amino]-2-carboxylic acid(24)

A solution of methyl N-2-methylbutyl-4-nitropyrrole-2-carboxylate 23(2.3 g, 0.98 mmol) in 20 ml of DMF was treated with a Pd/C catalyst(10%, 500 mg) and the mixture was hydrogenated in a Parr bom apparatus(500 psi H₂) for 7 hours. After 7 hours, Boc-anhydride (2.95 g, 13.5mmol) was added followed by DIEA (5 ml) and the reaction was stirredovernight. The reaction mixture was then partitioned between 200 ml ofwater and 200 ml of ethyl ether and extracted with ethyl ether (2×200ml). The combined organics were dried over sodium sulfate andconcentrated in vacuo to yield a yellow oil. The resulting yellow oilwas dissolved in 30 ml of methanol and 30 ml of 1M NaOH was added. Thesolution was heated at 50° C. for 6 hours, cooled to room temperature,extracted with ethyl ether (2×200 ml), acidified to pH 0 with sodiumbisulfate, and extracted with ethyl ether (3×200 ml). The combinedacidic extracts were dried (sodium sulfate) and concentrated in vacuo toyield a white solid. (1.2 g, 41% yield). ¹ H NMR (DMSO-d₆) δ 12.1 (br s,1 H), 9.09 (s, 1 H), 7.05 (s, 1 H), 6.59 (s, 1 H), 4.10 (d, 2H, J=7.1Hz), 3.35 (s, 2 H), 1.92 (m, 1 H), 1.44 (s, 9 H), 1.70 (d, 6 H, J=6.4Hz); ¹³ C NMR (DMSO-d₆) 175.0, 161.9, 112.9, 119.4, 118.5, 108.3, 108.2,55.0, 30.1, 28.4, 19.7.

Preparation of Monomers Substituted at the 3-Position of the PyrrolePreparation of 3-methyl Substituted Pyrroles

Scheme 14 outlines a general synthesis of a 3-methyl substitutedpyrrole. In this example the amine is protected with an allyloxycarbonylgroup. Briefly, diethylester (25) is methylated and hydrolyzed to yieldmonoacid 27. Monoacid 27 is reacted with allyl alcohol/DPPA to yieldallyl ester (28) which is hydrolyzed to form compound 29. ##STR22##

2, 4-Dicarbethoxy-1, 3-dimethylpyrrole (26)

A solution of 2, 4-dicarbethoxy-3 methylpyrrole (25) (2.44 g, 10.9mmol), K₂ CO₃ (9.9 g), and iodomethane (55 ml) in 750 ml acetone wasrefluxed at 50° C. for 15 hours. The solution was concentrated in vacuo,partitioned between 450 ml of dichloromethane and 600 ml of water. Theaqueous layer was extracted with CH₂ Cl₂ (2×500 ml), dried (sodiumsulfate), and concentrated in vacuo to yield a brown oil that solidifiedupon standing at room temperature for several minutes and was usedwithout further purification. (2.37 g, 91% yield). TLC (benzene) Rf 0.3;¹ H NMR (CD₂ Cl₂) δ 7.31 (s, 1H), 4.23 (m, 4H), 3.84 (s, 3H). 2.54 (2,3H), 1.33 (m, 6H).

1, 3-Dimethyl-4-carboxyl-2-carbethoxypyrrole (27)

2, 4-Dicarbethoxy-1, 3-dimethyl pyrrole (26) (9.24 g, 38.6 mmol) wassuspended in 120 ml of concentrated sulfuric acid and vigorously stirredfor 32 minutes at room temperature. The mixture was then precipitated bypouring into 2 liters of ice. The product was collected by vacuumfiltration, washed with water (5×250 ml), and dried in vacuo to providea white sand. (7.38 g, 91% yield). ¹ H NMR (CDCl₃) δ 7.42 (s, 1H), 4.35(m, 2H), 3.89 (s, 3H), 2.59 (s, 3H), 1.39 (t, 3H, J=7.1 Hz); ¹³ C NMR(CDCl₃) δ 237.1, 188.9, 184.4, 163.4, 161.6, 160.8, 159.9, 105.0, 46.6,15.6.

Preparation of Ethyl4-[(Allyloxycarbonyl)amino]-1,3-dimethylpyrrole-2-carboxylate (28)

1,3-dimethyl-4-carboxyl-2-carbethoxypyrrole (27) (2.19 g, 10.4 mmol),triethylamine (1.45 ml, 10.4 mmol) and diphenylphosphorylazide (DPPA)(Rappnport) (2.234 ml, 10.4 mmol) were dissolved in 31 ml of DNAsynthesis grade CH₃ CN (Fisher). The solution was refluxed for 4.5 hoursunder argon, after which allyl alcohol (31 ml) was added. The solutionwas refluxed for an additional 22 hours under argon. After 22 hours, thesolution was concentrated in vacuo, partitioned between 250 ml water and250 ml diethyl ether, washed several times with 10% Na₂ CO₃, 1M HCl, andwater. The organic layer separated, dried (sodium sulfate), andconcentrated to provide yellow crystals 28 (1.59 g, 58% yield). TLC(ethyl acetate) Rf 0.9; ¹ H NMR (DMSO) δ 8.77 (br, 1H), 7.05 (s, 1H)5.95 (m, 1H), 5.36 (d, 1H, J=17.1 Hz), 5.22 (d, 1H, J=10.4 Hz), 4.54 (d,2H, J=5.04 Hz), 4.19 (1, 2H, J=7.1 Hz), 3.75 (s, 3H), 2.102 (s, 3H),1.27 (t, 3H, J=7.1 Hz); ¹³ C NMR (DMSO) δ 161.3, 154.6, 133.8, 123.0,121.1, 117.4, 64.8, 59.4, 37.1, 14.5, 10.5.

Preparation of4-[(Allyloxycarbonyl)amino]-2-carboxyl-1,3-dimethylpyrrole (29)

Ethyl 4-[(allyloxycarbonyl)amino]-1,3-dimethylpyrrole-2-carboxylate (28)(1.00 g, 3.75 mmol) was suspended in 6 ml water. Methanol was added withvigorous stirring until all starting material dissolved 8 M NaOH (8 ml)was added, and the solution was stirred for five hours at 50° C. Thereaction mixture was allowed to cool to room temperature, and 1M HCladded to approximately pH 2 while solution cooled in ice bath toprecipitate out product. The product was collected by vacuum filtration,washed once with water, and dried in vacuo to yield 0.800 g (90% yield)of compound 29. TLC (ethyl acetate) Rf 0.8; ¹ H NMR (DMSO) δ 12.23 (br,1H), 8.73 (s, 1H), 7.01 (s, 1H), 5.93 (m, 1H), 5.33 (d, 1H, J=17.1 Hz),5.19 (d, 1H, J=10.5 Hz), 4.53 (d, 2H, J=5.5 Hz) 3.74 (s, 3H), 2.09 (s,3H); mass. spec. Calc. 238.0954 Found 238.0952.

Scheme 15 illustrates two syntheses of 3-hydroxy substituted pyrrolemonomers. Both syntheses utilize the previously described ethylN-methyl-2,4-carboxy-3-hydroxypyrrole (30) as a starting material(Momose et al. (1978) Chem. Pharm. Bull. 26:2224). In the firstapproach, the acid is converted to the allyl carbamate (31) using DPPAand allyl alcohol in a modified Curtius reaction. The hydroxy group isthen protected as a methyl ester with DMS, and the ethyl estersubsequently hydrolyzed with sodium hydroxide to yield compound (33).

The second approach produces a 3-substituted monomer which isappropriate as an N-terminal capping reagent. In this approach, ethylN-methyl-2,4-carboxy-3-hydroxypyrrole (30) is first decarboxylated underacidic conditions. The hydroxy group is then protected as the allylether, and the ethyl ester subsequently hydrolyzed to yield compound(36). ##STR23##

Preparation of Ethyl 4-allyloxycarbonyl-3-hydroxy pyrrole-2-carboxylate(31)

Ethyl N-methyl-2,4-carboxy-3-hydroxypyrrole (30) (500 mg, 2.36 mmol) wasdissolved in 7 ml of acetonitrile. Triethylamine (329 μl) was added tothis solution followed by DPPA (508 μl). The mixture was refluxed for1.5 hours, after which allyl alcohol was added (7.1 ml) and the mixturewas refluxed for an additional 17 hours. The reaction mixture was workedup using standard methods and the product was purified by flashchromatography (2% MeOH/CHCl₃ /AcOH) to yield 250 mg, (39%) of compound(31). FABMS (low res. 268 found, 268 calc.)

Preparation of 4-allyloxycarbonyl-3-methoxy pyrrole-2-carboxylic acid(33)

Compound 32 was prepared from Compound 31, using standard means (Dms,Na₂ CO₃) (Greene (1991) in Protecting Groups in Organic Synthesis, JohnWiley & Sons 2nd Ed., NY, N.Y.) To a solution of ethyl4-allyloxycarbonyl-3-methoxy pyrrole-2-carboxylate (32) (190 mg, 675μmol) in 12 ml of ethanol was added 0.1 sodium hydroxide (6.8 ml). Thesolution was refluxed for 3 days and worked up using standard methods.

Preparation of Ethyl 3-hydroxy pyrrole-2-carboxylate (34)

To a solution of ethyl N-methyl-2,4-carboxy-3-hydroxypyrrole (30) (1000mg, 4.7 mmol) in pyridine (8 ml) was added acetic acid (8 ml) and thesolution was refluxed for 6 hours. The reaction mixture was concentratedonto silica gel and purified by flash chromatography (25%) to yield 580mg (73%) of compound (34).

Preparation of Ethyl 3-allyloxy pyrrole-2-carboxylate (35)

To a solution of ethyl 3-hydroxypyrrole-2-carboxylate (34) (580 mg) inbenzene (12 ml) was added sodium hydride (387 mg). The suspension washeated at 60-70° C. for 1 hour. A solution of allyl bromide in benzenewas then added and the mixture heated at 70-80° C. for one hour. Thereaction was concentrated onto silica gel and purified by flashchromatography (25% EtOAc/hexane) to yield 300 mg (50%) of compound(35).

Example 2 Activation of Amino Acids

To activate the various amino acids 1.0 mmol of the appropriate aminoacid was dissolved in 2 ml DMF. HOBt (135 mg, 1.0 mmol) was addedfollowed by DCC (263 mg, 1 mmol) and the solution lightly shaken for atleast 30 minutes. The precipitated DCU by product was filtered beforeaddition to the coupling reaction.

Example 3 Preparation of Boc-Pyrrole-PAM and Boc Pyrrole-BAM-Resins (24)and (25) Preparation of Resin Linkage Agents 39 and 40

Resin linkage agents 39 and 40 were prepared in three steps according tothe published procedures of Merrifield, using Boc-Py-COOH as the aminoacid (Mitchell et al. (1978) J. Org. Chem. 43:2845-2852) as outlined inScheme 16. ##STR24##

Preparation of 4-(Bromomethyl)benzoic acid phenacyl ester (37)

Triethylamine (16 ml, 115 mmol) and bromoacetophenone (22.9 g, 115 mmol)were dissolved in 450 ml of ethyl acetate. The solution was stirred at50° C. and 4-(bromomethyl)benzoic acid (17.5 g, 155 mmol) was added inseven equal portions over a three hour period. Stirring was continuedfor an additional 8 hours at 50° C. Precipitatedtriethylaminehydrobromide was removed by filtration, and the ethylacetate solution was washed with (3×150 ml each) of 10% citric acid,brine, saturated sodium bicarbonate and brine. The organic phase wasdried with sodium sulfate and concentrated in vacuo. The residue wasrecrystallized from dichloromethane-petroleum ether to give fine whitecrystals (10.2 g, 27% yield). ¹ H NMR (DMSO-d₆) δ 7.99 (m, 4H),7.69-7.54 (m, 5H), 5.74 (s, 2H), 4.77 (s, 2H); ¹³ C NMR (DMSO-d₆) δ193.7, 165.9, 144.6, 134.9, 131.2, 131.0, 130.7, 130.6, 130.2, 129.9,128.8, 128.6, 68.2, 34.0.

Preparation of Boc-pyrrolyl-4-(oxymethyl)benzoic acid phenacyl ester(38)

A solution of Boc-pyrrole-OH (8)(2.9 g 12 mmol), 4-(bromomethyl)benzoicacid phenacyl ester (22) (4 g, 12 mmol) and diisopropylethylamine (3.0ml, 16.8 mmol) in 60 ml of DMF were stirred at 50° C. for 6 hours. Thesolution was cooled and partitioned between 400 ml of water and 400 mlof ethyl ether. The ether layer was washed with (2×200 ml each) of 10%citric acid, brine, saturated sodium bicarbonate and brine. The organicphase was dried with sodium sulfate and concentrated in vacuo to yieldcompound 38 as light white foam which was used without furtherpurification (5.4 g, 97% yield). TLC (2:3 hexane/ethyl acetate) Rf 0.6;¹ H NMR (DMSO-d₆) δ 9.14 (s, 1H), 8.03 (m, 4H), 7.67 (m, 1H), 7.55 (m,4H), 7.13 (s, 1H), 6.72 (d, 1H, J=1.5 Hz), 5.74 (s, 1H), 5.32 (s, 1H),3.79 (s, 3H), 1.42 (s, 9H); ¹³ C NMR (DMSO-d₆) δ 193.2, 165.5, 160.4,153.2, 143.1, 134.5, 130.1, 129.5, 128.3, 128.2, 123.8, 120.3, 118.8,108.2, 79.1, 67.7, 64.6, 36.7, 28.6.

Preparation of Boc-pyrrolyl-4-(oxymethyl)phenylacetic acid phenacylester

This compound was prepared by the method described above forBoc-pyrrolyl-4-(oxymethyl)benzoic acid phenacyl ester. The product waspurified by crystallization with hexane:ethyl acetate (3:1) as longneedles (6.1 g, 44.5%). TLC (3:1 hexane/ethyl acetate) Rf 0.2; ¹ H NMR(DMSO-d₆) δ 9.11 (s, 1H), 7.93 (d, 2H, J=8.2), 7.67 (t, 1H, J=7.0), 7.52(t, 2H, J=7.9), 7.35 (m, 4H), 7.10 (s, 1H), 6.67 (s, 1H), 5.50 (s, 2H),5.22 (s, 2H), 5.19 (s, 2H), 3.83 (s, 3H), 1.42 (s, 9H); ¹³ C NMR(DMSO-d₆) δ 193.1, 171.2, 160.6, 153.2, 135.7, 134.4, 130.1, 129.4,128.5, 128.3, 123.7, 120.0, 119.0, 108.0, 79.0, 67.4, 65.1, 36.7, 28.6.

Preparation of Boc-pyrrolyl-4-(oxymethyl)benzoic acid (39)

Boc-pyrrolyl-4-(oxymethyl)benzoic acid phenacyl ester (38) (3 g, 5.9mmol) was dissolved in 90 ml of acetic acid and water (80:20). Activatedzinc dust (9.6 g, 147 mmol) was added and the reaction was stirred for18 hours at room temperature. The zinc was removed by filtration and thereaction mixture was partitioned between 200 ml of ethyl ether and 200ml of water. The layers were separated and the aqueous layer wasextracted with another 200 ml ethyl ether. The ether layers werecombined and washed with (5×100 ml) of water. The combined organics weredried with sodium sulfate, concentrated in vacuo, and azeotroped with(6×100 ml) of benzene. The product was purified by flash chromatographywith a gradient of 2:1 hexane:ethyl acetate to ethyl acetate to give ayellow oil (1.9 g, 54%) of compound 39. TLC (ethyl acetate) Rf 0.7.

Preparation of Boc-pyrrolyl-4-(oxymethyl) phenylacetic acid (40)

Prepared in a manner analogous to 39, yielding 40 as a yellow oil in 78%yield.

Preparation of Boc-aminoacyl-pyrrolyl-4-(oxymethyl)-BAM-resin (41)

BAM linker acid (39) (1 g, 2.6 mmol) was dissolved in 6.5 ml of DMF/HOBt(382 mg, 2.8 mmol). DCC (735 mg, 2.8 mmol) was added and the reactionmixture was shaken at room temperature. After 4 hours the precipitatedDCU byproduct was filtered and the reaction mixture was added to 3 gramsaminomethyl-polystyrene-resin (0.7 mmol/gram substitution) previouslyswollen for 30 minutes in DMF. Diisopropylethylamine (913 μl, 5.3 mmol)was added and the reaction was shaken for 12 hours. After 12 hours theresin was determined by the ninhydrin test to be approximately 0.3mmol/gram substituted. At this time the resin was washed with DMF andthe remaining amine groups were capped by acetylation (2×) with excessacetic anhydride capping solution. The resin was washed with DMF,dichloromethane and MeOH and dried in vacuo.

Preparation of Boc-aminoacyl-pyrrolyl-4-(oxymethyl)-PAM-resin (42).Boc-Py-PAM-resin (42) (0.3 mmol/g substitution) was prepared using PAMlinker acid 40 as described above for the BAM resin.

EXAMPLE 4

Solid Phase Polyamide Synthesis

Preparation of Boc-PyPy-G-PyPyPy-G-PAM-resin. Boc-G-PAM-resin (1.25 g,0.25 mmol amine) was shaken in DMF for 15 minutes and drained. The N-bocgroup was removed by washing with dichloromethane for 1 minute, followedby washing with 65% TFA/CH₂ Cl₂ /0.5 M PhSH for 30 seconds, shaking in65% TFA/CH₂ Cl₂ /0.5 PhSH for 60 seconds, washing with 65% TFA/CH₂ Cl₂/PhSH for 30 seconds, and shaking in 65% TFA/CH₂ Cl₂ /PhSH for 20minutes. The trifluoroacetic acid deprotection mixture (65% TFA/CH₂ Cl₂/0.5 M PhSH) was prepared by combining and shaking a mixture oftrifluoroacetic acid (TFA) (290 ml), dichloromethane (150 ml), andthiophenol (23 ml, 225 mmol). The resin was washed for 1 minute withdichloromethane, 30 seconds with DMF, and shaken for 1 minute in DMF.The resin was then drained completely and activated acid, Boc-Py-OBt, (1mmol, 4 eq., prepared as described in Example 2) in 2 ml DMF was addedfollowed by DIEA (355 μl, 8 eq.) and the resin shaken vigorously to makea slurry. After shaking the reaction was allowed to proceed for 45minutes after which the reaction vessel was washed with DMF for 30seconds completing a single reaction cycle. Five additional cycles wereperformed adding, Boc-Py-OBt, Boc-Py-OBt, Boc-G-OBt, Boc-Py-OBt andBoc-Py-OBt to give Boc-PyPy-G-PyPyPy-G-PAM-Resin. The resin was washedwith DMF (1 minute), dichloromethane (1 minute) and methanol (1 minute)and dried in vacuo. This compound was then used to synthesizeImPyPy-G-PyPyPy-G-Ed (2a), ImPyPy-G-PyPyPy-G-Dp (2b),AcImPyPy-G-PyPyPy-G-Dp (2c), ImPyPy-G-PyPyPy-G-Ta-EDTA,ImPyPy-G-PyPyPy-G-Ta, and AcImPyPy-G-PyPyPy-G-Ta (2d) as describedbelow.

Preparation of AcImPyPy-G-PyPyPy-G-Dp (2c). A sample ofBoc-PyPy-G-PyPyPy-G-PAM-resin (600 mg, about 100 μmole) was placed in areaction vessel and shaken in DMF for 20 minutes. The resin wassubsequently drained and subjected to an additional coupling cycle withBoc-Im-OBt, as described above, to add an N-terminal Boc imidazole. TheN-Boc group was removed as described above and the resin was washed withdichloromethane (30 seconds) and DMF (1 minute). The resin was thentreated with 4 ml of an acetylation mixture (acetylation mixture: DMF (4ml), DIEA (710 μl, 4.0 mmol), and acetic anhydride (380 μl, 4.0 mmol)combined immediately before use) for 1 hour. The reaction vessel wasthen washed with DMF (1 minute), dichloromethane (1 minute) and methanol(1 minute) and dried in vacuo to yield AcImPyPy-G-PyPyPy-G-PAM-Resin.The resin (180 mg, 29 μmol) was weighed into a glass scintillation vialand treated with 1.5 ml of DMF, after 10 minutes, 1.5 ml ofdimethylaminopropylamine (Dp) was added and the mixture was shaken at37° C. for 12 hours. The resin was removed by filtration through anISOLAB polypropylene filter and washed with 11 ml of water. The DMFsolution and the water washes were combined. Seven milliliters of thecombined solution was loaded on a C₁₈ preparatory HPLC column, thecolumn was washed for 2 minutes in 0.1% TFA at 8 ml/min. to remove theDMF, followed by addition of a second 7 ml portion, which was alsowashed free of DMF with 0.1 % TFA for 2 minutes at 8 ml/min. No flushingof the polyamide occurs so long as the injection solution is less than20% v/v DMF. The polyamide was then eluted in 100 minutes with agradient of 0.25% CH₃ CN per minute. The polyamide was collected in 4-5separate 8 ml fractions, the purity of the individual fractions wasverified by HPLC and ¹ H NMR, to give AcImPyPy-G-PyPyPy-G-Dp (2c) (11.8mg, 39%).

Characterization of 2c. HPLC, r.t. 26.9; UV(H₂ O/DMSO)λ_(max) (ε), 246(45,200), 304 (50,200) (Extinction coefficients (ε) were determined bytaking a 5 μl aliquot from two separate NMR samples, diluting to 1 mlwith water and measuring the UV spectrum. The presence of 0.5% DMSO doesnot result in a significant change in the measured extinctioncoefficient.); ¹ H NMR (DMSO-d₆) δ 10.24 (s, 1H), 9.98 (s, 1H), 9.96 (s,1H), 9.94 (s, 1H), 9.92 (s, 1H), 9.90 (s, 1H), 9.2 (br s, 1H, CF₃ COOH),8.29 (m, 2H, G--NH and G--NH), 8.02 (t, 1H, J=6.6 Hz, PyCONH--G), 7.41(s, 1H), 7.26, (d, 1H, J=1.7 Hz), 7.23 (m, 3H), 7.16 (d, 1H, J =1.8 Hz),7.14 (d, 1H, J=1.7Hz), 7.05 (d, 1H J=1.8 Hz), 6.94 (m, 3H), 3.93 (s,3H), 3.89 (d, 2H, J=3.9 Hz, Gly CH₂), 3.84 (s, 3H), 3.84 (s, 3H), 3.83(s, 3H), 3.80 (s, 3H), 3.79 (s, 3H), 3.72 (d, 2H, J=4.9 Hz, GlyCH₂),3.14 (q, 2H, J=5.0 Hz), 3.03 (q, 2H, J=6.0 Hz), 2.74 (d, 6H, J=6.0 Hz,CH₂ N(CH₃)₂), 2.00 (s, 3H, CH₃ CONH), 1.77 (quintet, 2H, J=4.6 Hz, CH₂CH₂ N(CH₃)₂); MALDI-TOF MS, calcd M⁺ H 993.1, found 993.8.

Preparation of ImPyPy-G-PyPyPy-G-Dp (2b). A sample ofBoc-PyPy-G-PyPyPy-G-PAM-Resin (600 mg, about 100 μmole) was placed in areaction vessel and shaken in DMF for 15 minutes. The N-Boc group wasremoved with TFA as described above and the resin was washed withdichloromethane (30 seconds) and DMF (1 minute) and was treated with theHOBt ester of N-methyl imidazole-2-carboxylic acid (about 16equivalents, prepared as described below) and DIEA (155 μl, 16 eq.) for2 hours. The resin was washed with DMF, dichloromethane and MeOH (1minute each) and dried in vacuo to yield ImPyPy-G-PyPyPy-G-PAM-Resin. Itshould be noted that polyamides capped with N-methylimidazole-2-carboxylic acid tend to give false positives for the picricacid test even when reactions are >99% complete as determined bystepwise HPLC analysis. The crude product was cleaved from the resin(180 mg, 29 μmole) with dimethylaminopropylamine and purified asdescribed for (2c) to yield ImPyPy-G-PyPyPy-G-Dp (2b) (12 mg, 40%recovery).

Characterization of 2b. HPLC, r.t. 26.9; UV(H₂ O/DMSO)λ_(max) (ε), 246(41,100), 304 (48,400); ¹ H NMR (DMSO-d₆) δ 10.49 (s, 1H), 9.98 (s, 1H),9.95 (s, 1H), 9.92 (s, 1H), 9.89 (s, 1H), 9.2 (br s, 1H, CF₃ COOH), 8.30(m, 2H, Gly-NH and Gly-NH), 8.06 (t, 1H, J=5.8 Hz, PyCONH-Gly), 7.40 (s,1H), 7.24, (d, 1H, J=1.7 Hz), 7.23 (m, 3H), 7.17 (m, 2H), 7.06 (m, 2H),6.94 (m, 3H), 3.99 (s, 3H), 3.89 (d, 2H, Gly CH₂), 3.84 (s, 3H), 3.84(s, 3H), 3.83 (s, 3H), 3.81 (s, 3H), 3.80 (s, 3H), 3.72 (d, 2H, J=4.3Hz, GlyCH₂), 3.13 (q, 2H, J=5.7 Hz), 3.01 (q, 2H, J=5.2 Hz), 2.76 (d,6H, J=4.3 Hz, CH₂ N(CH₃)₂), 1.77 (quintet, 2H, J=7.4 Hz, CH₂ CH₂N(CH₃)₂); MALDI-TOF MS, calc. for (M⁺ H⁺) 936.0, found 935.7.

Preparation of ImPyPy-G-PyPyPy-G-Ed (2a). ImPyPy-G-PyPyPy-G-PAM-Resin(180 mg, 29 μmole, synthesized as described for 2b was shaken in 1.5 mlof DMF. After 20 minutes, ethylenediamine (Ed) (1.5 ml) was added andthe mixture was shaken at 37° C. for 12 hours. The crude product waspurified as described for 2c to yield ImPyPy-G-PyPyPy-G-Ed (2a) (9 mg,39%).

Characterization of 2a. HPLC, r.t. 24.2; V(H₂ O/DMSO)λ_(max) (ε), 246(44,400), 304 (51,300); ¹ H NMR (DMSO-d₆) δ 10.49 (s, 1H), 9.97 (s, 1H),9.94 (s, 1H), 9.92 (s, 1H), 9.88 (s, 1H), 8.30 (t, 1H, J=2.6 Hz), 8.23(t, 1H, J=5.6 Hz), 8.04 (t, 1H, J=3.1 Hz), 7.73 (br s, 1H), 7.40 (s,1H), 7.28 (d, 1H, J=1.7 Hz), 7.23 (d, 1H, J=1.7 Hz), 7.22 (m, 2H), 7.15(m, 2H), 7.05 (m, 2H), 6.95 (m, 3H), 3.98 (s, 3H), 3.88 (d, 2, J=4.1),3.83 (s, 3H), 3.82 (m, 6H), 3.80 (s, 3H), 3.79, (s, 3H), 3.76 (m, 2H),3.29 (m, 2H), 2.84 (m, 2H); MALDI-TOF MS, calc. for (M⁺ H⁺) 893.9, found894.9.

Preparation of AcImPyPy-G-PyPyPy-G-Ta (2d).AcImPyPy-G-PyPyPy-G-PAM-Resin (180 mg, 29 μmole, synthesized asdescribed for 2c) was treated with 1.5 ml of DMF. After 20 minutes, 1.5ml of 3,3'-diamino-N-methylpropylamine (Ta) was added and the mixturewas shaken at 37° C. for 12 hours and purified as described for (2c) toyield AcImPyPy-G-PyPyPy-G-Ta (2d) (6.7 mg, 23% yield).

Characterization of 2d. HPLC, r.t. 25.9; UV(H₂ O/DMSO)λ_(max) (ε), 246(43,600), 304 (51,800); ¹ H NMR (DMSO-d₆) δ 10.23 (s, 1H), 9.99 (s, 1H),9.96 (s, 1H), 9.94 (s, 1H), 9.91 (s, 1H), 9.89 (s, 1H) 9.53 (br s, 1H,CF₃ COOH), 8.28 (m, 2H, J=6.1 Hz, Gly-NH and Gly-NH), 8.04 (t, 1H, J=5.3Hz, PyCONH-Gly), 7.79-7.82 (br s, 3H, CH₂ NH₃), 7.41, (s, 1H), 7.25 (d,1H, J=1.7 Hz), 7.23 (d, 1H, J=1.7 Hz), 7.22 (d, 1H, J=1.7 Hz), 7.20 (d,2H, J=1.7 Hz), 7.15 (d, 1H, J=1.7 Hz), 7.13 (d, 1H, J=1.7 Hz), 7.05 (d,1H, J=1.7 Hz), 6.94 (m, 2H), 6.92 (d, 1H, J=1.7 Hz), 3.89 (s, 3H), 3.88(d, 2H, GlyCH₂), 3.85 (s, 3H), 3.84 (s, 3H), 3.82 (s, 3H), 3.80 (s, 3H),3.79 (s, 3H), 3.71 (d, 2H, J=5.5 Hz, GlyCH₂), 3.37 (m, 2H), 3.13 (m,4H), 2.80 (m, 2H), 2.73 (d, 3H, J=3.3 Hz, NCH₃), 2.01 (s, 3H, CH₃ CO),1.89 (m, 2H), 1.77 (m, 2H); MALDI-TOF MS, calc. for (M⁺ H⁺) 1036.2,found 1036.2.

AcImPyPy-G-PyPyPy-G-Ta-EDTA (2e). To a solution ofAcImPyPy-G-PyPyPy-G-PAM-resin (synthesized as described for 2c) (3.0 mg,2.5 μmole) in 750 μl of DMSO was added 750 μl NMP, followed by EDTAmonoanhydride (30 mg, 118 μmole) and the solution was heated at 37° C.After 2 hours, 13 ml of water was added and the reaction was purified bypreparatory HPLC as described above. The EDTA derivative eluted at 120minutes to give AcImPyPy-G-PyPyPy-G-Ta-EDTA (2e) (1.1 mg, 32% yield).

Characterization of 2e. HPLC, r.t. 27.8; UV(H₂ O/DMSO)λ_(max), 246, 304;¹ H NMR (DMSO-d₆) δ 10.23 (s, 1H), 9.99 (s, 1H), 9.96 (s, 1H), 9.94 (s,1H), 9.91 (s, 1H), 9.89 (s, 1H) 9.25 (br s), 8.43 (t, 1H), 8.33 (m, 2H),8.06 (t, 1H) 7.41, (s, 1H), 7.26 (d, 1H, J=1.7 Hz), 7.22 (d, 1H, J=1.7Hz), 7.21 (d, 1H, J=1.7 Hz), 7.20 (d, 2H, J=1.7 Hz), 7.15 (d, 1H, J=1.7Hz), 7.13 (d, 1H, J=1.7 Hz), 7.07 (d, 1H, J=1.7 Hz), 6.94 (m, 2H), 6.92(d, 1H, J=1.7), 3.93 (s, 3H), 3.88 (d, 2H), 3.85 (s, 3H), 3.84 (s, 3H),3.82 (s, 3H), 3.80 (s, 3H), 3.79 (s, 3H), 3.71 (d, 2H), 3.65 (m 4H),3.26 (m, 1OH), 3.13 (m, 4H), 2.71 (d, 3H), 2.00 (s, 3H) 1.78 (m, 6H),1.21 (m, 2H); MALDI-TOF MS, calc. for (M⁺ H⁺) 1310.4, found 1311.7.

Preparation of ImPyPyPyPyPyPy-G-Ed (1a). This compound was synthesizedand purified by the general procedures described above to yield 6.7 mg(19%) of ImPyPyPyPyPyPy-G-Ed (la) which was 97% pure. A portion of thismaterial was purified a second time by preparatory HPLC to give pure 1a(0.8 mg).

Characterization of 1a. HPLC, r.t. 28.3; UV(H₂ O/DMSO)λ_(max) (ε), 246(35,600), 312 (57,000); ¹ H NMR (DMSO-d₆) δ 10.48 (s, 1H), 9.99 (s, 1H,PyNH), 9.96 (m, 4H, PyNH), 8.26 (t, 1H, J=6.3 Hz, G--NH), 8.04 (t, 1H,J=5.3 Hz, PyCONH-Gly), 7.75-7.67 (br s, 3H, NH₃), 7.40 (s, 1H), 7.28 (d,1H J=1.7 Hz), 7.23 (m, 5H), 7.17 (d, 1H, J=1.7 Hz), 7.08 (m, 4H), 7.05(d, 1H, J=1.7 Hz), 6.95 (d, 1H, J=1.7 Hz), 3.98 (s, 3H), 3.85 (m, 15 H),3.79 (s, 3H), 3.74 (d, 2H, J=6.4 Hz, GlyCH₂), 3.31 (q, 2H, J=6.1 Hz,Gly-NH--CH₂), 2.85 (m, 2H, J=3.0 Hz, CH₂ NH₂); MALDI-TOF MS, calc. forM⁺ H⁺ 959.0, found 959.3.

Preparation of ImPyPyPyPyPyPy-G-Dp (1b). This compound was synthesizedby the general procedures described above to give 8 mg, 24% yield ofImPyPyPyPyPyPy-G-Dp (1b). A portion of this material was purified asecond time by preparatory HPLC to give pure 1b (1.2 mg).

Characterization of 1b. HPLC, r.t. 28.5; UV(H₂ O/DMSO)λ_(max) (ε), 246(34,600), 312 (55,300); ¹ H NMR (DMSO-d₆) δ 10.55 (s, 1H, PyNH), 10.02(s, 1H, PyNH), 10.00 (m, 4H, PyNH), 9.3 (br s, 1H, CF₃ COOH), 8.32 (t,1H, J=6.2 Hz, Gly-NH), 8.06 (t, 1H, J=5.9 Hz, PyCONH-Gly), 7.44 (d, 1H,J=0.6 Hz), 7.31 (d, 1H, J=1.7 Hz), 7.26 (m, 5H), 7.19 (d, 1H, J=1.8 Hz),7.10 (m, 5H), 6.97 (d, 1H, J=1.7 Hz), 4.01 (s, 3H), 3.87 (m, 15 H), 3.82(s, 3H), 3.73 (d, 2H, J=5.5 Hz, GlyCH₂), 3.16 (q, 2H, J=6.2 Hz,Gly-NH--CH₂), 3.03 (q, 2H, J=5.2 Hz, CH₂ N(CH₃)₂), 2.74 (d, 6H, J=4.9Hz, CH₂ N(CH₃)₂), 1.77 (quintet, 2H, J=6.7 Hz, CH₂ CH₂ N(CH₃)₂);MALDI-TOF MS, calc. for 1001.1, found 1000.5.

Preparation of ImPyPyPyPyPyPy-G-Ta (1c). This compound was synthesizedby the general procedures described above to yield 9.2 mg, (28%) ofproduct 1c.

Characterization of 1e. HPLC, r.t. 29.3; UV(H₂ O/DMSO)λ_(max) (ε), 246(33, 400), 312 (53,500), ¹ H NMR (DMSO-d₆) δ 10.47 (s, 1H, PyNH), 9.95(m, 5H, PyNH), 9.4 (br s, 1H, CF₃ COOH), 8.28 (t, 1H, J=6.1 Hz, Gly-NH),8.04 (t, 1H, J=5.1 Hz, PyCONH-Gly), 7.8 (br s, 3H, CH₂ NH₃), 7.39, (d,1H, J=0.6 Hz), 7.28 (d, 1H, J=1.2 Hz), 7.23 (m, 5H), 7.17 (d, 1H, J=1.8Hz), 7.09 (m, 4H), 7.04 (d, 1H, J=1.7 Hz), 6.96 (d, 1H, J=1.6 Hz), 3.98(s, 3H), 3.85 (m, 15 H), 3.79 (s, 3H), 3.72 (d, 2H, J=5.2 Hz, GlyCH₂),3.15 (q, 2H, J=5.0 Hz), 3.11 (m, 4H), 2.80 (m, 2H), 2.74 (d, 3H, J=2.9Hz, NCH₃), 1.89 (quintet, 2H, J=7.4 Hz), 1.77 (quintet, 2H, J=6.8 Hz);MALDI-TOF MS, calc. for M⁺ H⁺ 1044.2, found 1044.1.

Preparation of ImPyPyPyPyPyPy-G-Ta-EDTA (1d). Synthesized by the generalprocedures described above to yield 1.1 mg, 32% yield of compound 1d.

Characterization of 1d. HPLC, r.t. 30.6; UV(H₂ O/DMSO)λ_(max), 246, 312;¹ H NMR (DMSO-d₆) δ 10.47 (s, 1H), 9.46 (m, 4H), 7.39 (s, 1H), 7.28 (d,1H, J=1.7 Hz), 7.28 (d, 1H, J=1.6 Hz), 7.24 (d, 1H, J=1.6 Hz), 7.23 (m,4H), 7.17 (d, 1H, J=1.7 Hz), 7.08 (m, 5H), 7.04 (d, 1H, J=1.6 Hz), 6.95(d, 1H, J=1.5 Hz), 3.98 (s, 3H), 3.84 (m, l5H), 3.79 (s, 3H), 3.71 (d,2H), 3.66 (m, 4H), 3.26 (m, 8H), 3.13 (m, 4H), 2.73 (d, 3H), 2.27 (t,2H), 1.78 (m, 6H), 1.21 (m, 2H); MALDI-TOF MS, calc. for M⁺ H⁺ 1317.4,found 1318.1.

Preparation of AcImPyPy-γ-PyPyPy-G-Dp (3a). This compound wassynthesized by the general procedures set forth above to yield 13.1 mg(30%) of compound 3a. The only variation was that Boc-γ is activated insite

Characterization of 3a. HPLC, r.t. 24.0; UV(H₂ O/DMSO)λ_(max) (ε), 246(35,900), 312 (48,800); ¹ H NMR (DMSO-d₆) δ 10.23 (s, 1H), 9.98 (s, 1H),9.32 (s, 1H), 9.90 (m, 2H), 9.84 (s, 1H), 9.2 (br s, 1H), 8.27 (t, 1HJ=5.0 Hz), 8.05 (m, 2H), 7.41 (s, 1H), 7.25 (d, 1H J=1.2 Hz), 7.22 (m,2H), 7.16 (m, 2H), 7.12 (d, 1H, J=1.7), 7.05 (d, 1H, J=1.5 Hz), 6.94 (d,1H, J=1.6 Hz), 6.89 (d, 1H, J=1.7 Hz) 6.87 (d, 1H, J=1.6Hz), 3.93 (s,3H), 3.83 (s, 3H), 3.82 (m, 6H), 3.81 (s, 3H), 3.79 (s, 3H), 3.71 (d,2H, J=5.1 Hz), 3.19 (m, 2H, J=5.8), 3.12 (m, 2H J=5.0Hz), 3.01 (m, 2H,J=4.2 Hz), 2.74 (d, 6H, J=4.6 Hz), 2.26 (m 2H, J=4.6 Hz), 2.00 (s, 3H),1.75 (m, 4H); MALDI-TOF MS, calc. for M⁺ H⁺ 1021.1, found 1021.6.

Preparation of AcImPyPy-γ-PyPyPy-G-Ta (3b). This compound wassynthesized by the general procedures set forth above to yield 9.2 mg(31%) of product 3b.

Characterization of 3b. HPLC, r.t. 24.9; UV(H₂ O/DMSO)λ_(max) (ε), 246(37,400), 312 (50,500);¹ H NMR (DMSO-d₆) δ 10.24 (s, 1H), 9.98 (s, 1H),9.94 (s, 1H), 9.91 (m, 2H), 9.85 (s, 1H), 9.7 (br s, 1H), 8.28 (t, 1H,J=5.2Hz), 8.05 (m, 2H), 7.86 (br s, 3H), 7.42 (d, 1H, J=1.6 Hz), 7.26(d, 1H, J=1.7Hz), 7.22 (m, 2H), 7.17 (m, 2H), 7.13 (d, 1H, J=1.7 Hz),7.06 (d, 1H, J=1.7 Hz), 6.95 (d, 1H, J=1.7 Hz), 6.90 (d, 1H, J=1.7Hz),6.88 (d, 1H, J=1.7 Hz), 3.94 (s, 3H), 3.84 (s, 6H), 3.82 (s, 3H), 3.80(s, 6H), 3.72 (s, 3H), 3.14 (m, 6H), 3.08 (m, 2H), 2.85 (m, 2H), 2.75(d, 3H, J=4.2 Hz), 2.72 (t, 2H, J=6.8Hz), 2.01 (s, 3H), 1.90 (m, 2H),1.75 (m, 4H); MALDI-TOF-MS, calc. for M⁺ H⁺ 1064.2, found 1064.5.

Preparation of AcImPyPy-γ-PyPyPy-G-Ta-EDTA (3c). Synthesized by thegeneral procedures described above to yield 3.2 mg (41%) of compound 3c.

Characterization of 3c. HPLC, r.t. 24.3; UV(H₂ O/DMSO)λ_(max), 246,312;¹ H NMR (DMSO-d₆) δ 10.32 (s, 1H), 9.97 (s, 1H), 9.93 (s, 1H), 9.90 (m,2H), 9.84 (s, 1H), 8.40 (t, 1H), 8.27 (t, 1H), 8.05 (m, 2H), 7.41 (s,1H), 7.25 (d, 1H, J=1.6 Hz), 7.20 (m, 2H), 7.16 (m, 2H), 7.11 (d, 1H,J=1.6 Hz), 7.05 (d, 1H, J=1.7 Hz), 6.94 (d, 1H, J=1.7 Hz), 6.86 (d, 1H,J=1.7 Hz), 3.92 (s, 3H), 3.83 (s, 3H), 3.82 (s, 3H), 3.81 (s, 3H), 3.78(m, 6H), 3.72 (d, 2H, J=J=5.5 Hz), 3.66 (m, 4H), 3.40 (m, 1OH), 3.15 (m,6H), 2.73 (d, 3H, J=4.2 Hz), 2.27 (t, 2H, J=6.9 Hz), 2.03 (s, 3H), 1.78(m, 6H), 1.23 (m, 2H); MALDI-TOF-MS, calc. for M⁺ H⁺ 1339.4, found 1340.

Preparation of ImImPy-γ-PyPyPy-G-Dp (4d). Synthesized by the generalprocedures described above to yield 8.9 mg (30%) ImImPy-γ-PyPyPy-G-Dp(4d).

Characterization of 4d. HPLC, r.t. 24.6; UV(H₂ O/DMSO)λ_(max) (ε), 246(37,600), 312 (50,700); ¹ H NMR (DMSO-d₆) δ 10.30 (s, 1H), 10.28 (s,1H), 9.93 (s, 1H), 9.90 (s, 1H), 9.84 (s, 1H), 9.33 (s, 1H), 9.28 (br s, 1H), 8.05 (t, 1H, J=5.1), 8.08 (t, 1H), 8.02 (t, 1H), 7.56 (s, 1H).7.50 (s, 1H), 7.21 (m, 3H), 7.16 (d, 1H, J=1.2 Hz), 7.05 (d, 1H, J=1.1Hz), 7.00 (d, 1H, J=1.4 Hz), 6.94 (d, 1H, J=1.4 Hz), 6.87 (d, 1H, J=1.2Hz), 3.99 (s, 3H), 3.97 (s, 3H), 3.83 (s, 3H), 3.82 (s, 3H), 3.80 (s,3H), 3.79 (s, 3H), 3.71 (d, 2H, J=4.2 Hz), 3.20 (m, 2H), 3.14 (m, 2H),3.01 (m, 2H,), 2.75 (d, 6H, J=3.2 Hz), 2.27 (t, 2H, J=7.2 Hz), 2.03 (s,3H), 1.76 (m, 4H); IR (neat) 3260 (m), 2927 (w) 2332 (w), 1666 (s), 1531(s), 1449 (m), 1396 (w), 1196 (w), 1126 (w); MALDI-TOF-MS, calc. for C₄₇H₆₀ N₁₈ O₉ M⁺ H⁺ 1022.1, found 1022.4.

AcImImPy-γ-PyPyPy-G-Dp (4a). Synthesized by the general proceduresdescribed above to yield 8.9 mg (30%) of compound 4a.

Characterization of 4a. HPLC, r.t. 24. 1; UV(H₂ O/DMSO)λ_(max) (ε), 246(37,600), 312 (50,700); ¹ H NMR (DMSO-d₆) δ 10.30 (s, 1H), 10.28 (s,1H), 9.93 (s, 1H), 9.90 (s, 1H), 9.84 (s, 1H), 9.33 (s, 1H), 9.28 (br s,1H), 8.05 (t, 1H, J=5.1 Hz), 8.08 (t, 1H), 8.02 (t, 1H), 7.56 (s, 1H),7.50 (s, 1H), 7.21 (m, 3H), 7.16 (d, 1H, J=1.2 Hz), 7.05 (d, 1H, J=1.1Hz), 7.00 (d, 1H, J=1.4 Hz), 6.94 (d, 1H, J=1.4 Hz), 6.87 (d, 1H, J=1.2Hz), 3.99 (s, 3H), 3.97 (s, 3H), 3.83 (s, 3H), 3.82 (s, 3H), 3.80 (s,3H), 3.79 (s, 3H), 3.71 (d, 2H, J=4.2 Hz), 3.20 (m, 2H), 3.14 (m, 2H),3.01 (m, 2H, J=6.2 Hz), 2.75 (d, 6H, J=3.2 Hz), 2.27 (t, 2H, J=7.2 Hz),2.03 (s, 3H), 1.76 (m, 4H); MALDI-TOF-MS, calc. for M⁺ H⁺ 1022.1, found1022.7.

AcImImPy-γ-PyPyPy-G-Ta (4b). Synthesized by the general proceduresdescribed above to yield 7.4 mg (25%) of compound 4b.

Characterization of 4b. HPLC, r.t. 23.8; UV(H₂ O/DMSO)λ_(max) (ε), 246(37,000), 312 (50000); ¹ H NMR (DMSO-d₆) δ 10.31 (s, 1H), 10.29 (s, 1H),9.93 (s, 1H), 9.90 (s, 1H), 9.84 (s, 1H), 9.34 (s, 1H), 8.31 (t, 1H,J=5.0 Hz), 8.08 (m, 2H), 7.80 (br s, 3H), 7.56 (s, 1H), 7.50 (s, 1H),7.20 (m, 3H), 7.15 (d, 1H, J=1.2 Hz), 7.06 (d, 1H, J=1.2 Hz), 7.00 (d,1H, J=1.3 Hz), 6.95 (d, 1H, J=1.2 Hz), 6.88 (d, 1H, J=1.3 Hz), 3.98 (s,3H), 3.96 (s, 3H), 3.82 (s, 3H), 3.82 (s, 3H), 3.79 (m, 6H), 3.71 (d,2H, J=4.9 Hz), 3.15 (m, 6H), 3.06 (m, 2H, J=4.7 Hz), 2.84 (m, 2H, J=4.9Hz), 2.74 (d, 3H, J=4.2 Hz), 2.27 (t, 2H), 2.02 (s, 3H), 1.89 (m, 2H),1.75 (m, 4H); MALDI-TOF-MS, calc. for M⁺ H⁺ 1065.2, found 1065.2.

Preparation of AcImImPy-γ-PyPyPy-G-Ta-EDTA (4c). Synthesized by thegeneral procedures described above to yield 2.1 mg (35%) of product 4c.

Characterization of 4c. HPLC, r.t. 23.8; UV(H₂ O/DMSO)λ_(max), 246, 312;¹ H NMR (DMSO-d₆) δ 10.31 (s, 1H), 10.29 (s, 1H), 9.93 (s, 1H), 9.90 (s,1H), 9.84 (s, 1H), 9.33 (s, 1H), 9.21 (br s, 1H), 8.37 (t, 2H, J=4.6Hz), 8.28 (t, 1H, J=5.2 Hz), 8.09 (t, 1H, J=5.2 Hz), 8.03 (t, 1H, J=5.4Hz), 7.6 (s, 1H), 7.5 (s, 1H), 7.21 (m, 3H), 7.15 (d, 1H, J=0.8 Hz),7.06 (d, 1H, J=1.0 Hz), 7.00 (d, 1H, J=1.2 Hz), 6.95 (d, 1H, J=1.1 Hz),6.88 (d, 1H, J=1.2 Hz), 3.99 (s, 3H), 3.96 (s, 3H), 3.83 (s, 3H), 3.82(s, 3H), 3.79 (m, 6H), 3.71 (d, 2H, J=5.0Hz), 3.66 (m, 4H), 3.25 (m,1OH), 3.15 (m, 6H), 2.72 (d, 3H, J=4.4 Hz), 2.47 (t, 2H), 2.00 (s, 3H),1.77 (m, 6H), 1.27 (m, 2H); MALDI-TOF-MS, calc. for M⁺ H⁺ 1338.4, found1338.6.

Preparation of AcPyPyPy-γ-ImImPy-Gly-Dp (5a). Synthesized by the generalprocedures described above to yield 9.9 mg (37%) of compound 5a.

Characterization of 5a. HPLC, r.t. 23.8; UV(H₂ O/DMSO)λ_(max) (ε), 246(41,800), 312 (56,400); ¹ H NMR (DMSO-d₆) δ 10.34 (s, 1H), 10.33 (s,1H), 9.90 (m, 1H), 9.83 (s, 1H), 9.35 (s, 1H), 9.29 (br s, 1H), 8.29 (t,1H, J=J=5.3 Hz), 8.03 (m, 2H), 7.56 (s, 1H), 7.53 (s, 1H), 7.27 (d, 1H,J=1.0Hz), 7.22 (d, 1H, J=7.16Hz), 7.16 (d, 1H, J=0.9Hz), 7.13 (d, 1H,J=1.0 Hz), 7.03 (m, 2H), 6.88 (d, 1H, J=1.1 Hz), 6.84 (d, 1H, J=1.0 Hz),3.98 (s, 3H), 3.97 (s, 3H), 3.92 (s, 3H), 3.83 (s, 3H), 3.81 (s, 3H),3.79 (s, 3H), 3.72 (s, 3H), Gly CH₂ was covered by water, 3.20 (m, 2H,J=5.6 Hz), 3.12 (m, 2H, J=5.9 Hz), 3.02 (quintet, 2H, J=4.1 Hz), 2.74(d, 6H, J=4.4 Hz), 2.36 (t, 2H, J=7.0 Hz), 1.95 (s, 3H), 1.77 (m, 4H);MALDI-TOF-MS, calc. for M⁺ H⁺ 1022.1, found 1022.4.

Preparation of AcPyPyPy-γ-ImImPy-G-Ta (5b). Synthesized by the generalprocedures described above to yield 8.2 mg (27%) of compound 5b.

Characterization of 5b. HPLC, r.t. 23.6; UV(H₂ O/DMSO)λ_(max) (ε), 246(39,300), 312 (53,100); ¹ H NMR (DMSO-₆) δ 10.38 (s, 1H), 10.34 (s, 1H),9.92 (m, 2H), 9.85 (s, 1H), 9.35 (s, 1H), 8.33 (t, 1H), 8.07 (m, 2H),7.82 (br s, 1H), 7.57 (s, 1H), 7.54 (s, 1H), 7.28 (d, 1H, J=1 Hz), 7.23(d, 1H, J=1Hz), 7.17 (d, 1H, J=Hz), 7.14 (d, 1H, J=1Hz), 7.14 (d, 1H,J=1.3Hz), 7.04 (m, 2H), 6.89 (d, 1H, J=I Hz), 6.84 (d, 1H, J=1 Hz), 3.99(s, 3H), 3.97 (s, 3H), 3.83 (s, 3H), 3.82 (s, 3H), 3.81 (s, 3H), 3.79(s, 3H), 3.71 (d, 2H, J=5.2 Hz), 3.26 (m, 2H), 3.14 (d, 2H, J=5.1 Hz),3.05 (m, 2H), 2.83 (q, 2H, J=5.6 Hz), 2.74 (d, 3H, J=4.3 Hz), 2.39 (m,2H), 1.96 (s, 3H), 1.88 (q, 2H, J=6.6 Hz), 1.78 (m, 2H); MALDI-TOF-MS,calc. for M⁺ H⁺ 1065.2, found 1065.9.

Preparation of AcPyPyPy-γ-ImImPy-G-Ta-EDTA (5c). Synthesized by thegeneral procedures described above to yield 3.1 mg (37%) of 5c.

Characterization of 5c. HPLC, r.t. 24.0; UV(H₂ O/DMSO)λ_(max) (ε), 246,312; ¹ H NMR (DMSO-d₆) δ 10.37 (s, 1H), 10.34 (s, 1H), 9.91 (m, 2H),9.84 (s, 1H), 9.37 (s, 1H), 8.38 (t, 1H), 8.32 (t, 1H), 8.06 (m, 2H),7.57 (s, 1H), 7.53 (s, 1H), 7.27 (s, 1H), 7.22 (s, 1H), 7.17 (s, 1H),7.14 (s, 1H), 7.04 (m, 2H), 6.88 (s, 1H), 6.85 (s, 1H), 3.99 (s, 3H),3.96 (s, 3H), 3.83 (s, 3H), 3.82 (s, 3H), 3.79 (m, 6H), 3.71 (d, 2H),3.64 (m, 4H), 3.25 (m, 1OH), 3.15 (m, 6H), 2.72 (d, 3H), 2.50 (t, 2H),1.95 (s, 3H), 1.79 (m, 6H), 1.22 (m, 2H); MALDI-TOF-MS, calc. for M⁺ H⁺1338.4, found 1339.1.

Preparation of EDTA-γ-ImPyPy-β-PyPyPy-G-Dp. The polyamideImPyPy-β-PyPyPy-G-Dp, synthesized by the general procedures describedabove, was modified with the dianhydride of EDTA in DMSO/NMP at 55° C.for 10 minutes, the anhydride was then opened with 0.1 M NaOH (20minutes), and the reaction mixture purified by reverse phase preparatoryHPLC to provide the EDTA modified polyamide.

Preparation of ImPyPy-G-PyPyPy-β-Dp. The polyamide was prepared asdescribed above to yield a white powder. (12.3 mg, 42% recovery). HPLC,r.t. 25.5; UV(H₂ O/DMSO)λ_(max) (ε), 246 (39,500), 312 (52,000) nm; ¹ HNMR (DMSO-d₆) δ 10.46 (s, 1H), 9.96 (s, 1H), 9.90 (s, 1H), 9.88 (m, 2H),9.21 (br s, 1H) 8.27 (t, 1H, J=452.5 Hz), 8.06 (m, 2H), 7.39 (s, 1H),7.28 (d, 1 H, J=1.6 Hz), 7.23 (d, 1H, J=1.7 Hz), 7.20 (d, 1H, J=1.5 Hz),7.15 (m, 3H), 7.04, (m, 2H), 7.03 (d, 1H, J=1.6 Hz), 6.94 (d, 1H, J=1.7Hz), 6.92 (d, 1H, J=1.4 Hz), 3.98 (s, 3H), 3.88 (d, 2H), 3.83 (s, 3H),3.82 (m, 6H), 3.79 (s, 3H), 3.78 (s, 3H), 3.36 (q, 2H, J=5.3 Hz), 3.09(q, 2H, J=6.0 Hz), 2.99 (m, 2H), 2.75 (t, 2H, J=5.2 Hz), 2.72 (d, 6H,J=4.8 Hz), 2.30 (t, 2H, J=6.3 Hz), 1.72 (quintet, 2H, J=5.7 Hz);MALDI-TOF MS 950.06; FABMS m/e 949.462 (M+H 949.455 calc. for C₄₅ H₅₇N₁₆ O₈).

EXAMPLE 5

Symmetric Anhydride Activation of Pyrrole. In a typical symmetricanhydride procedure (0.25 mmol synthesis cycle), the resin was washedwith 5% DIEA/CH₂ Cl₂. No DIEA should be present, however, at the startof the coupling reaction. Boc-pyrrole-COOH (514 mg, 2 mmol) was slurriedin 3 ml dichloromethane, DCC (406 mg, 2 mmol) was then added upon whichtime the white slurry turned clear. After three minutes,dimethylaminopyridine (DMAP) (101 mg, 1 mmol) was added and the solutionwas stirred, filtered and added to the reaction vessel containing theresin. The coupling was allowed to proceed for 2 hours, 355 μl DIEA wasthen added and the reaction allowed to proceed for an additional hour.

EXAMPLE 6

Picric Acid Test. In order to monitor the progress of the reactions 8-10mg samples were periodically removed from the deprotection reactionmixtures and evaluated using picric acid titration. The 10 mg sample waswashed with dichloromethane, 5% TEA/CH₂ Cl₂, and dichloromethane, anddried either at 50° C. or by aspiration. A sample of about 5 mg of thedried resin was weighed into a disposable polypropylene filter,successively washed using gravity filtration with approximately: 5 mldichloromethane, 5 ml 0.1 M picric acid/dichloromethane and 50 mldichloromethane to carefully remove any excess picric acid, the picricacid salt eluted with 5% DIEA/CH₂ Cl₂ (3×500 μl). The DIEA/CH₂ Cl₂ washwas collected and diluted with 4 ml MeOH and the absorbance measured at358 nm.

EXAMPLE 7

Stepwise HPLC analysis. Approximately 2 mg of a resin sample was placedin a 1.5 ml polypropylene tube, 40 μl of DMF was added and the resin wasallowed to stand for 10 minutes. 40 μl of dimethylaminopropylamine wasthen added and the mixture was vortexed, briefly centrifuged, and heatedat 37° C. for 12 hours. After 12 hours the solutions were againvortexed, centrifuged, and a 10 μl aliquot was taken, diluted with 90 μlwater and analyzed by analytical HPLC under standard conditions at 254nm.

EXAMPLE 8

Cleavage of the Polyamide from the Resin using Pd(OAc)₂. Scheme 17illustrates a general method for cleaving the synthesized polyamide fromthe resin. The acetylated tripyrrole AcPyPyPy-PAM-resin is used forpurposes of illustration. ##STR25##

The PAM or BAM pyrrole resin was treated with Pd(OAc)₂ in DMF under apressurized atmosphere of hydrogen (100 psi, 8 hours). The palladiumblack was filtered and the pyrrole acid activated with DCC/HOBt andreacted with a large excess of dimethylaminopropylamine to give the HPLCpurified acetylated tripyrrole in 5% overall yield. HPLC and NMR areconsistent with that of an authentic standard synthesized by solutionphase methods by Wade et al. (1992) J. Am. Chem. Soc. 114:8783 8794.

EXAMPLE 9

Cleavage of the Polyamide from the G-PAM-resin. Scheme 18 illustrates ageneral method for cleaving the synthesized polyamide from theG-PAM-resin. The acetylated tripyrrole AcPyPyPy-PAM-G-PAM-resin is usedfor purposes of illustration. ##STR26##

180 mg (29 μmole) of AcPyPyPy-PAM-G-PAM-resin was treated with 1.5 mlDMF followed by 1.5 ml dimethylaminopropylamine and the reaction mixtureshaken for 12 hours, and purified by preparatory HPLC to giveAcPyPyPy-PAM-G-Dp in 49% yield. ¹ H NMR (DMSO-d₆) δ 9.90 (m, 2H), 9.83(s, 1H), 9.3 (br s, 1H), 8.37 (t, 1H, J=5.7 Hz), 8.05 (t, 1H, J=5.8 Hz),7.44 (d, 1H, J=1.7 Hz), 7.32 (q, 4H, J=8.2 Hz), 7.20 (d, 1H, J=1.7 Hz),7.13 (d, 1H, J=1.7Hz), 7.04 (d, 1H, J=1.7Hz), 6.95 (d, 1H, J=1.9Hz),6.83 (d, 1H, J=1.8Hz), 5.19 (s, 2H), 3.82 (s, 3H), 3.82 (s, 3H), 3.81(s, 3H), 3.6 (d, 2H, J=6.1 Hz), 3.48 (s, 2H), 3.11 (q, 2H, J=6.1 Hz),2.96 (m, 2H), 2.67 (d, 6H, J=4.8Hz), 1.95 (s, 3H), 1.71 (quintet, 2H,J=7.4Hz). A failure sequence was also isolated from the reaction mixturein 25% yield. ¹ H NMR (DMSO-d₆) δ 9.91 (m, 2H), 9.80 (s, 1H), 9.3 (br s,1H), 8.40 (t, 1H, J=5.7 Hz), 8.08 (t, 1H, J=5.8 Hz), 7.44 (d, 1H, J=1.7Hz), 7.38 (q, 4H, J=8.4Hz), 7.15 (d, 1H, J=1.7 Hz), 6.96 (d, 1H,J=1.8Hz), 6.85 (d, 1H, J=1.7Hz), 5.15 (s, 2H), 3.84 (s, 3H), 3.82 (s,3H), 3.69 (d, 2H, J=5.4 Hz), 3.51 (s, 2H), 3.19 (m, 2H), 3.04 (m, 2H),2.74 (d, 6H, J=4.2 Hz), 1.97 (s, 3H), 1.77 (m, 2H).

EXAMPLE 10

Quantitative DNase I footprint titrations. All reactions were executedin a total volume of 40 μL. A polyamide stock solution (H₂ O containingno polyamide was used for reference reactions) was added to an assaybuffer containing radio labeled restriction fragment (15,000 cpm),affording final solution conditions of 10 mM Tris HCl, 10 mM KCl, 10 mMMgCl₂, 5 mM CaCl₂, pH 7.0 and (i) 0.1 nM-1 μM polyamide, for allpolyamides except ImPyPy-β-PyPyPy-Dp and ImPyPy-β-PyPyPy-G-Dp, (ii).0.01 nM-0.1 μM polyamide for ImPyPy-β-PyPyPy-Dp andImPyPy-β-PyPyPy-G-Dp. The solutions were allowed to equilibrate for 5hours at 22° C. Footprinting reactions were initiated by the addition of4 μL of DNase I stock solution (at the appropriate concentration to give55% intact labeled DNA) containing 1 mM dithiothreitol. The reactionswere allowed to proceed for approximately seven minutes at 22° C. Afterseven minutes the reactions were by the addition of 10 μL of a solutioncontaining 1.25 M NaCl, 100 mM EDTA, and 0.2 mg/ml glycogen, and ethanolprecipitated. The reactions were resuspended in 1×TBE/80% formamideloading buffer, denatured at 85° C. for 10 minutes, placed on ice, andloaded onto an 8% polyacrylamide gel (5% cross-link, 7 M urea). Thereaction products were separated by electrophoresis in 1×TBE at 2000 V.Gels were dried and exposed to a storage phosphor screen (MolecularDynamics). FIG. 15 depicts the storage phosphor autoradiogram of 8%denaturing polyacrylamide gels used to separate the fragments generatedby DNase I digestion in quantitative footprint titration experiments:lanes 1-2, A and G sequencing lanes; lanes 3 and 21, DNase I digestionproducts obtained in the absence of polyamide; lanes 4-20, DNase Idigestion products obtained in the presence of 0.1 nM (0.01 nM), 0.2 nM,(0.02 nM), 0.5 nM (0.05 nM), 1 nM (0.1 nM), 1.5 nM (0.15 nM), 2.5 nM(0.25 nM), 4 nM (0.4 nM), 6.5 nM (0.65 nM), 10 nM (1 nM), 15 nM (1.5),25 nM (2.5 nM), 40 nM (4 nM), 65 nM (6.5 nM), 100 nM (10 nM), 200 nM (20nM), 500 nM (10 nM), 1 μM (0.1 μM) concentrations were used forpolyamides ImPyPy-β-Ala-PyPyPy-Dp and ImPyPy-β-Ala-PyPyPy-Dp only are inparentheses); lane 22, intact DNA. The five binding sites that wereanalyzed by quantitative footprint titration experiments are indicatedon the right sides of the autoradiogram.

Data from the footprint titration gels were obtained using a MolecularDynamics 400S PhosphorImager followed by quantitation using ImageQuantsoftware (molecular Dynamics).

Background-corrected volume integration of rectangles encompassing thefootprint sites and a reference site at which DNase I reactivity wasinvariant across the titration generated values for the site intensities(I_(site)) and the reference intensity (I_(ref)). The apparentfractional occupancy (q_(app)) of the sites were calculated using theequation: ##EQU1## where I°_(site) and I°_(ref) are the site andreference intensities, respectively, from a control lane to which nopolyamide was added.

The ([L]_(tot), θ_(app)) data points were fit to a general Hill equation(eq) by minimizing the difference between θ_(app) and θ_(fit) : ##EQU2##where [L]_(tot) is the total polyamide concentration, K_(a) is theapparent first-order association constant, and θ_(min) and θ_(max) arethe experimentally determined site saturation values when the site isunoccupied or saturated, respectively. The data were fit using anonlinear least-squares fitting procedure with K_(a), n, θ_(min) as theadjustable parameters. In cases for which the best-fit value of n was≦1.5, the data were fit with n=2, with K_(a), θ_(max), and θ_(min) asthe adjustable parameters. The binding isotherms were normalized usingthe following equation: ##EQU3## Three sets of data were used indetermining each association constant.

At higher concentrations of polyamide (>˜0.1 μM for ImPyPy-β-PyPyPy-Dpand ImPyPy-β-PyPyPy-Dp, and >1 μM for the other six polyamides), thereference sites become partially protected due to non-specificDNA-binding, resulting in low θ_(app) values. For this reason, higherconcentrations were not used. As a consequence, association constantsfor sites that are not saturated or nearly saturated at the highestconcentration of polyamide used can be determined only approximately.The method for determining association constants used here involved theassumption that [L]_(tot) =[L]_(free) where [L]_(free) is theconcentration of polyamide free in solution (unbound). For very highassociation constants this assumption becomes invalid, resulting inunderestimated association constants. In these experiments, theconcentration of DNA is estimated to be 50 pM. As a consequence,association constants of 2×10⁹ M⁻¹ and 5×10⁹ M⁻¹ will be underestimatedby approximately 90% and 80%, respectively.

EXAMPLE 11

Preparation of Dimers

Preparation of Ethyl4-[[(tert-butyloxy)carbonyl]amino]-1-methylpyrrole-2-(4-carboxamido-1-methyl-imidazole)-2-carboxylate(44). To a solution of4-[[(tert-butyloxy)carbonyl]amino]-1-methylpyrrole-2-carboxylic acid (8g, 33 mmol) in 20 ml DMF was added 1.2 eq HOBt (5.3 g, 39 mmol) followedby 1.2 eq DCC (8 g, 39 mmol). The solution was stirred for 24 hours,after which the DCU byproduct was removed by filtration. Separately, toa solution of ethyl 4-nitro-1-methylimidazole-2-carboxylate (8 g, 40mmol) in 20 ml DMF was added Pd/C catalyst (10%, 1 g), and the mixturehydrogenated in a Parr bom apparatus (500 psi H₂) for 2 hours. Thecatalyst was removed by filtration through celite, and the filtrateimmediately added to the -OBt ester solution, an excess of DIEA (10 ml,110 mmol) added, and the mixture stirred at 37° C. for 48 hours. Thereaction mixture was added dropwise to a stirred solution of ice waterand the resulting precipitate collected by vacuum filtration and driedin vacuo to yield a brown powder. (12.3 g, 94% yield). ¹ H NMR (DMSO-d₆)δ 10.7 (s, 1H), 9.2 (s, 1H), 7.6 (s, 1H), 6.8 (d, 1H), 4.3 (q, 2H), 3.9(s, 3H), 3.7 (s, 3H), 1.5 (s, 9H), 1.3 (t, 3H).

Preparation of4-[[(tert-butyloxy)carbonyl]amino]-1-methylpyrrole-2-(4-carboxamido-1-methylimidazole)-2-carboxylicacid (45). To a solution of ethyl4[[tert-butyloxy)carbonyl]amino]-1-methylpyrrole-2-(4-carboxamido-1-methylimidazole)-2-carboxylate(44) (5 g, 12.7 mmol) in 50 ml methanol was added 50 ml 1 M KOH and thereaction was allowed to stir for 6 hours at 37° C. Excess methanol wasremoved in vacuo and the resulting solution acidified by the addition of10% potassium bisulfate. The resulting precipitate was collected byvacuum filtration and dried in vacuo to yield a brown powder. (4.4 g,89% yield). ¹ H NMR (DMSO-d₆) δ 10.9 (s, 1H), 8.9 (s, 1H), 7.6 (s, 1H),7.3 (d, 1H), 6.9 (d, 1H), 4.1 (s, 3H), 3.9 (s, 3H), 1.4 (s, 9H).

Preparation of Ethyl γ-[[tert-butyloxy)carbonyl]amino]-butyric acid-(4-carboxamido-1-methylimidazole)-2-carboxylate (46). To a solution ofBoc-γ-aminobutyric acid (10 g, 49 mmol) in 40 ml DMF was added 1.2 eqHOBt (7.9 g, 59 mmol) followed by 1.2 eq DCC (11.9 g, 59 mmol). Thesolution was stirred for 24 hours, and the DCU byproduct was removed byfiltration. Separately, to a solution of ethyl4-nitro-1-methylimidazole-2-carboxylate (9.8 g, 49 mmol) in 20 ml DMFwas added Pd/C catalyst (10%, 1 g), and the mixture was hydrogenated ina Parr bom apparatus (500 psi H₂) for 2 hours. The catalyst was removedby filtration through celite and the filtrate was immediately added tothe -OBt ester solution. An excess of DIEA (15 ml) was then added andthe reaction was stirred at 37° C. After 48 hours the reaction mixturewas added dropwise to a stirred solution of ice water and the resultingprecipitate collected by vacuum filtration and dried in vacuo to yield abrown powder. (9.4 g, 54% yield). ¹ H NMR (DMSO-d₆) δ 10.6 (s, 1H), 7.6(s, 1H), 6.9 (t, 1H), 4.2 (q, 2H), 3.9 (s, 3H), 2.9 (q, 2H), 2.3 (t,2H), 1.4 (s, 9H), 1.3 (t, 3H).

Preparation of γ-[[(tert-butyloxy)carbonyl]amino]-butyricacid-(4-carboxamido-1-methyl-pyrrole)-2-carboxylate (47). To a solutionof ethyl γ-[[(tert-butyloxy)carbonyl]amino]-butyricacid-(4-carboxamido-1-methyl)-2-carboxylate (5 g, 14.1 mmol) in 50 mlmethanol was added 50 ml 1 M KOH and the resulting mixture was allowedto stir for 6 hours at 37° C. Excess methanol was removed in vacuo andthe resulting solution acidified by the addition of 10% potassiumbisulfate. The resulting precipitate was collected by vacuum filtrationand dried in vacuo to yield a brown powder. (4.1 g, 91% yield). ¹ H NMR(DMSO-d₆) δ 10.6 (s, 1H), 7.6 (s, 1H), 6.8 (t, 1H), 3.9 (s, 3H), 2.8 (q,2H), 2.3 (q, 2H), 1.7 (t, 2H), 1.5 (s, 9H).

Activation of Boc-X-Im-COOH. Boc-γ-Im-COOH or Boc-Py-Im-COOH (100 mg,about 0.3 mmol) and HBTU (2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluoro phosphate) (118 mg, 0.3 mmol) were dissolved in 500μl DMF, 100 μl DIEA was added and the solution allowed to stand for 3minutes.

EXAMPLE 12

Preparation of Allyl Protected Monomers

Scheme 19 illustrates a general method for the preparation of allylprotected monomers for use in preparation of cyclic polyamides.##STR27##

Methyl-4-nitropyrrole-2-carboxylic acid (49). To a solution of sodiumnitrite (714 g, 10.2 mol) in 700 ml of water at 50° C.±5° C. was addedover a period of 3 hours a solution of mucobromic acid (48) (700 g, 2.7mol) in 700 ml of warm ethanol. The reaction was stirred for 15 minutes,cooled to 0° C., and 700 ml of ethanol was added. The resulting orangeprecipitate was collected by vacuum filtration and dried in vacuo toyield sodium nitromalondialdehyde monohydrate (305 g, 1.9 mol) which wasused in the next step without further purification. Glycine ethyl ester(300 g, 2.9 mol) and nitromalondialdehyde monohydrate (305 g, 1.9 mol)were placed in a 12 l round bottom flask and slurried with a mechanicalstirrer in 560 ml of methanol and 280 ml of water. Sodium hydroxide (800g, 20 mol) dissolved in 1.6 l water was added to the solution at a ratewhich maintained the temperature at 50° C. The reaction was cooled to 0°C. with an ice bath and neutralized to pH 0 with 1.7 1 HCl (conc.) whilemaintaining the temperature below 10° C. A black precipitate was removedby filtration through Celite and the product was extracted with 20liters of ethyl acetate. The organic layer was dried with sodium sulfateand concentrated in vacuo to provide 4-nitropyrrole-2-carboxylic acid(140 g, 0.9 mol) as a brown solid which was used without furtherpurification. To a solution of 4-nitropyrrole-2-carboxylic acid (140 g,0.9 mol) dissolved in 180 ml of methanol was added 8 ml of concentratedsulfuric acid. The solution was refluxed for 15 hours, cooled to 0° C.and 90 ml of water was added. The solution was then allowed to stand at-20° C. for 2 days. The resulting light brown crystals were collected byvacuum filtration to yield pure methyl 4-nitropyrrole-2-carboxylate (49)(105 g, 0.62 mol) in 23% overall yield. ¹ H NMR (DMSO-d₆) δ 14.2 (br s,1H), 7.74 (d, 1H, J=1.6 Hz), 7.35 (d, 1H, J=1.7 Hz), 3.73 (s, 3H).

Preparation of 1-(Benzyloxycarbonylmethyl)-2-carboxy-4-nitropyrrolemethyl ester (50). To a solution of methyl 4-nitropyrrole-2-carboxylate(49) (8.1 g, 48 mmol) dissolved in 100 ml of acetone was added potassiumcarbonate (19.5 g, 141 mmol) and potassium iodide (7.2 g, 43.4 mmol),followed by 2-benzylbromoacetate (18.9 ml). The solution was refluxedfor 2 hours, an additional 5 ml of 2-benzylbromoacetate was added andthe solution was refluxed for an additional 2 hours. The reactionmixture was concentrated in vacuo, partitioned between 300 ml of waterand 300 ml of dichloromethane and extracted with dichloromethane (2×100ml). The extracts were combined, dried over sodium sulfate andconcentrated in vacuo. The resulting oil was purified by flashchromatography (4:1 hexane:ethyl acetate) to yield 910.4 g (32.7 mmol,68%) of the diester (50). ¹ H NMR (DMSO-d₆) δ 8.30 (d, 1H, J=1.9 Hz),7.35 (d, 1H, J=1.9 Hz), 7.35 (s, 5H), 5.27 (s, 2H), 5.19 (s, 2H), 3.17(s, 3H); ¹³ C NMR (DMSO-d₆) δ 168.1, 160.3, 135.2, 130.2, 129.0, 128.8,128.6, 123.1, 112.1., 67.1, 52.5, 51.5; FABMS, found 318.085, calc.318.085.

Preparation of methyl1-(carboxymethyl)-4-[[tert-butyloxy)carbonyl]amino]-2-carboxylate (51).To a solution of diester (50) (4.3 g, 13.5 mmol) in 80 ml of DMF wasadded Pd/C catalyst (10%, 1 g) and the mixture was hydrogenated in aParr bom apparatus (500 psi H₂) for 7 hours. Boc-anhydride (2.95 g, 13.5mmol) was then added followed by DIEA (6 ml, 66 mmol) and the reactionwas stirred for 2 hours. The Pd/C catalyst was removed by filtrationthrough Celite and the reaction mixture was partitioned between 500 mlof bicarbonate and 500 ml of dichloromethane and extracted withdichloromethane (2×200 ml). The pH was then reduced to three with 10%citric acid and the mixture was extracted with dichloromethane (4×200ml.) The combined acidic extracts were dried (sodium sulfate) andconcentrated in vacuo to yield a brown oil. The crude mixture waspurified by flash chromatography (10% MeOH/dichloromethane) to yield awhite solid. (2.8 g, 69.5% yield). ¹ H NMR (DMSO-d₆) δ 12.75 (br s, 1H),9.15 (s, 1H), 7.14 (s, 1H), 6.64 (2, 1H), 4.90 (s, 2H), 3.68 (s, 3H),1.46 (s, 9H).

Preparation of allyl1-(carboxymethyl)-4-[[(tert-Butyloxy)carbonyl]amino]-2-carboxylate (52).To a solution of the Boc-methyl ester (51) (500 mg, 1.6 mmol) in 5 ml ofdry allyl alcohol was added a solution of 60% sodium hydride (640 mg)dissolved in 10 ml of allyl alcohol. The gel-like mixture was refluxedfor 30 minutes and cooled to room temperature. 100 ml of 10% citric acidwas added and the reaction mixture was extracted with dichloromethane(3×200 ml). The combined organics were dried and concentrated in vacuoto yield the pure Boc allyl ester (31) as a brown oil. (408 mg, 82%yield). ¹ H NMR (DMSO-d₆) δ 9.18 (s, 1H), 7.18 (s, 1H), 6.68 (s, 1H),5.91-6.01 (m, 1H), 5.21-5.34 (dd, 2H), 4.96 (s, 2H), 4.3 (d, 2H, J=5.2Hz), 1.45 (s, 9H); ¹³ C NMR (DMSO-d₆) δ 170.4, 160.0, 152.9, 133.1,123.4, 119.7, 117.8, 107.9, 78.8, 64.0, 50.0, 28.3; FABMS found 313.140,calc. 313.140.

EXAMPLE 13

Preparation of cyclo-(-γ-ImPyImPy-γ-ImPyImPy(-G-Dp)-) (57)

Synthesis of the linear precursor H₂ N-γ-ImPyImPy-γ-ImPyImPy(-G-Dp)-COOH(56). Boc-G-PAM resin (1.25 g, 0.25 mmol) was deprotected under standardconditions. Boc allyl monomer 52 (101 mg, 0.325 mmol) and HOBt (88 mg,0.65 mmol) were dissolved in 400 μl DMF and DCC (66 mg, 0.325 mmol) wasadded. After 15 minutes DCU was removed by filtration and the activatedester was added to the reaction vessel, followed by 1.5 ml DMF and 355μl DIEA. The reaction was allowed to proceed for 12 hours providingBoc-Py(O-allyl)-G-resin (53). The remaining polyamide was synthesized bystandard solid phase methods of this invention to provide H₂N-γ-ImPyImPy-γ-ImPyImPy(O-allyl)-G-resin (54). To remove the allylprotecting group, the resin was then treated with THF (2×200 ?? ml) and10 ml of a solution of 593 μl n-butylamine and 250 μl formic acid in 25ml THF was then added, followed by 280 mg Pd₂ (dba)₃ -CHCl₃ and 980 mgtriphenylphosphine. The reaction mixture was shaken for 3 hours at roomtemperature, drained, rinsed with acetone (200 ml), 0.1 M sodiumN,N-diethyl-dithiocarbamate in water (50 ml, 2×1 min), acetone (200 ml),water (200 ml), 0.1 M sodium N,N-diethyl-dithiocarbamate in water (50ml, 2×1 min), acetone (200 ml), water (200 ml), methanol (200 ml),dichloromethane (200 ml), methanol (200 ml), and the resin was dried invacuo. The polyamide was then cleaved and purified under standardconditions to yield the linear precursor (56) as a fluffy white solid.(105 mg, 45% yield). HPLC, r.t. 27.0 min.; ¹ H NMR (DMSO-d₆) δ 10.37 (s,1H), 10.29 (s, 1H), 10.23 (s, 1H), 10.22 (s, 1H), 10.18 (s, 1H), 9.96(s, 1H), 9.92 (s, 1H), 9.86 (s, 1H), 9.31 (br s, 1H), 8.31 (t, 1H), 8.06(t, 1H), 7.85 (t, 1H), 7.71 (br s, 3H), 7.53 (m, 2H), 7.44 (s, 1H), 7.36(s, 1H), 7.189 (d, 1H, J=1.5 Hz), 7.12 (d, 1H, J=1.7 Hz), 6.97 (d, 1H,J=1.6 Hz), 6.94 (d, 1H, J=1.5 Hz), 4.97 (s, 2H), 3.96 (m, 6H), 3.94 (m,9H), 3.85 (m, 6H), 3.79 (s, 3H), 3.66 (d, 1H, J=5.7 Hz), 3.23 (m, 4H),2.98 (m, 2H), 2.79 (m, 2H), 2.71 (d, 6H, J=4.4 Hz), 2.33 (m, 4 Hz), 1.77(m, 6H); MALDI-TOF MS, 1355.4, found 1355.7 calc.

Removal of the allyl group yielded no undesirable side products asdetermined by HPLC.

Synthesis of cyclo-(-γ-ImPyImPy-γ-ImPyImPy(-G-Dp)- (57). The linearprecursor (56) (16 mg) was dissolved in 7 ml of DMF, DPPA (28 mg) wasadded followed by potassium carbonate (45 mg) and the reaction mixturerapidly shaken for 3 hours, upon which time the reaction was determinedto be complete by HPLC analysis r.t. 29 min. The cyclic peptide waspurified by preparatory HPLC. MALDI-TOF MS 1355.4 calc., 1355.7 found(FIG. 19).

EXAMPLE 14

Preparation of Oligonucleotide-Polyamide Conjugates

Materials. The following additional materials are needed for synthesisof oligonucleotide-polyamide conjugates.6-(4-monomethoxytritylamino)propyl-(2-cyanoethyl)-(N,N-diisopropyl)phosphoramidite,5'-amino-modifier C6,12-(4-monomethoxytritylamino)propyl-(2-cyanoethyl)-(N,N-diisopropyl)phosphoramidite,5'-amino-modifier C12, dT CE (2-cyanoethyl) phosphoramidite, 0.45 Msublimed tetrazole in acetonitrile, THF/lutidine/Ac₂ O (8:1:1), 10% MeImin THF, 0.1 M I₂ in THF/Pyridine/H₂ O, 3% TCA/CH₂ Cl₂, 5-methylcytidineCE (2-cyanoethyl)phosphoramidite, and bulk 500 Å dT-Icaa-CPG werepurchased from Glen Research. All 10 μmole preparation columns werepacked manually from bulk support.1,2,3-benzotriazol-1-yl-4[[(tert-butyloxy)carbonyl]-amino]-1-methylpyrrole-2-carboxylateand1,2,3-benzotriazol-1-yl-4[[(tert-butyloxy)carbonyl]-amino]-1-methylimidazole-2-carboxylatewere prepared as previously described. (Baird and Dervan Manuscript inPreparation; Schnozler et al. (1992) Int. J. Pep. Prot. Res. 40:180-193;Grehn and Ragnarsson (1981 J. Org. Chem. 46:3492-3497; Grehn et al.(1990) Acta. Chem. Scand. 44:67-74.). 0.0002 M potassiumcyanide/pyridine, and acetic anhydride (Ac₂ O) were purchased fromApplied Biosystems. HPLC analysis was performed either on a HP 1090 Manalytical HPLC or a Beckman Gold system using a RAINEN C18, MicrosorbMV, 5 μm, 300×4.6 mm reversed phase column in 100 mM ammonium acetate,pH 4.9 with acetonitrile as eluent and a flow rate of 1.0 ml/min,gradient elution 1.0% acetonitrile/min. Oligonucleotide conjugates werepurified by FPLC (Pharmacia) on a ProRPC HR 10/10 reversed phase columnusing a linear gradient from 0 to 40% acetonitrile in 55 minutes, 100 mMtriethylammonium acetate, pH 7.0.

Preparation of ImPyPy-CONH(CH₂)₆ --P(O)₄ TTTTTT^(m) C^(m) CTTT-3' (62)(SEQ ID NO:7). The oligonucleotide DMT-TTTTTT^(m) C^(m) CTTT-CPG wasprepared on an Applied Biosystems Model 394B DNA synthesizer using amanually prepared 10 μmole synthesis column and a standard 10 μmolesynthesis cycle. C₆ -Aminomodifier-MMT (100 μmole) was dissolved in1,100 μl of anhydrous acetonitrile, vortexed vigorously, and placed onthe synthesizer. The amino modifier was added by machine synthesis usinga modified 10 μmole synthesis cycle with an extended 10 minute couplingtime and the MMT group left on. The column was manually washed with 50ml of 3% trichloroacetic acid/dichloromethane until a yellow color wasno longer observed in the wash (approximately 12 minutes). The columnwas then washed with 15 ml dichloromethane and dried in vacuo. The CPGwas transferred to a 5 ml glass manual peptide reaction vessel andwashed with DMF (30 seconds). A sample was taken for ninhydrin test andan absorbance consistent with 50 μmole/gram substitution was found.

Boc-pyrrole-OBt ester (9) (70 mg, 200 μmole) was dissolved in DMF (600μl) and DIEA (68 μl) was added. The coupling mixture was added to thereaction vessel and the mixture was shaken for 60 minutes. The resin waswashed with DMF (30 seconds), dichloromethane (30 seconds) and 65%TFA/CH₂ Cl₂ /0.5 M PhSH (30 seconds). The resin was shaken in 65%TFA/CH₂ Cl₂ /0.5 M PhSH for 20 minutes, drained, washed withdichloromethane (30 seconds) followed by DMF (30 seconds). A secondequivalent of Boc-pyrrole-OBt was added under identical conditions tothe first, and the reaction shaken for 1 hour, washed with DMF (30seconds), dichloromethane (30 seconds) and treated with 65% TFA/CH₂ Cl₂/0.5 M PhSH as described for the first deprotection.N-methylimidazole-2-carboxylic acid (133 mg) was activated in 1 ml ofDMF with HOBt/DCC as described above and added to the reaction vesselwith DIEA (200 μl) and the reaction was shaken to shake for 2 hours. TheCPG was washed with DMF (30 seconds), dichloromethane (30 seconds) anddried in vacuo. The entire CPG (approximately 210 mg) was placed in 2 mlof 0.1 M NaOH and heated at 55° C. for 15 hours. The CPG was removed byfiltration through a polypropylene filter, and 1 ml of 1 Mtriethylammonium acetate, pH 7, was added and the reaction diluted to 10ml total volume with water. The mixture was purified in two separateportions by FPLC. In each run the conjugate was triple injected in 1.5ml volume portions. Collected fractions were lyophilized, andrelyophilized from water to yield the desired conjugateImPyPy-CONH(CH₂)₆ P(O)₄ -TTTTTT^(m) C^(m) CTTT-3' (62) (SEQ ID NO:7) (15mg, 37% yield).

Characterization of ImPyPy-CONH(CH₂)₆ P(O)₄ -TTTTTT^(m) C^(m) CTTT-3'(62). Analytic HPLC (10 nmole), r.t., 14.8 min.; UV(H₂ O/DMSO)λ_(max)(ε), 260 (118,000), 304 (33,000); 18,800 u, Enzymatic digestion (10nmole), 7.8 min. (mC, 2441 u), 9.0 min. (T, 15,235 u) 26.2 min.(peptide, 4257 u (260), 1843 u 340); ¹ H NMR (D₂ O) δ 7.71 (s, 1H), 7.69(s, 1H), 7.50 (m, 9H), 7.19 (s, 1H), 7.18 (s, 1H), 7.12 (d, 1H, J=1.2Hz), 7.05 (d, 1H, J=1.3Hz), 6.89 (d, 1H, J=1.3Hz), 6.67 (d, 1H,J=1.3Hz), 6.11 (m, 11H), 4.89, 4.76, 4.70, 4.22, 4.01, 3.91, 3.88, 3.75,3.42, 3.11, 2.40, 2.25, 2.20, 1.80, 1.75, 1.41, 1.23; MALDI-TOF MS,calc. M-1 3814.3, found 3813.5.

Synthesis of AcImPyPy-CONH(CH₂)₁₂ P(O)₄ -TTTTTT^(m) C^(m) CTTT-3' (63)(SEQ ID NO:8). Boc-PyPy-CONH(CH₂)₁₂ P(O)₄ -TTTTTT^(m) C^(m) CTTT-CPG wasassembled as described above for compound 62. The N-boc group wasremoved with 65% TFA/CH₂ Cl₂ /0.5 M PhSH and the resin was treated witha solution of Boc-Im-OBt (13) (70 mg), DIEA (68 μl) and DMF (600 μl),and shaken for 1 hour. The CPG was washed with DMF (30 seconds),dichloromethane (30 seconds) and dried in vacuo. One third of the CPG,70 mg, was removed from the synthesis and placed in a 10 μmole DNAsynthesis column. Two syringes were used simultaneously to manipulatereagents into and out of the column. The column was washed withdichloromethane and carefully treated with 65% TFA/CH₂ Cl₂ /0.5 M PhSHto remove the N-Boc group. The column was carefully washed withdichloromethane (20 ml) and DMF (20 ml) and then treated withacetylation mixture for 1 hour, washed with DMF (20 ml) and CH₂ Cl₂ (20ml) and the cartridge dried in vacuo. The resin was removed from thecolumn and placed in 1 ml 0.1 M NaOH at 55° C. for 15 hours. The CPG wasremoved by filtration, 1 ml of 1 M pH 7 triethylammonium acetate wasadded and the mixture purified by FPLC. The appropriate fractions werecollected and concentrated in vacuo to give AcImPyPy-CONH(CH₂)₁₂ P(O)₄-TTTTTT^(m) C^(m) CTTT-3' (63) (SEQ ID NO:8) (336 nmole, 10% yield).

Characterization of AcImPyPy-CONH(CH₂)₁₂ P(O)₄ -TTTTTT^(m) C^(m) CTTT-3'(63). Analytic HPLC (10 nmole), r.t., 19.7 min.; UV(H₂ O/DMSO)λ_(max)(ε), 260 (110,000), 304 (36,000); 13,000 u, Enzymatic digestion (10nmole), 7.7 min. (mC, 1551 u), 8.9 min. (T, 1 1374 u), 33.2 min.(peptide, 1646 u (260), 1281 u 340); MALDI-TOF MS, calc. M-1 3953.5,found 3952.9.

Preparation of Boc-γ-ImPyPy-CONH(CH₂)₁₂ P(O)₄ -TTTTTT^(m) C^(m) CTTT-3'(64) (SEQ ID NO:9). A sample of Boc-ImPyPy-CONH(CH₂)₁₂ P(O)₄ -TTTTTT^(m)C^(m) CTTT-CPG (140 mg), prepared by the general procedures describedabove was placed in a 10 μmole DNA synthesis column. The column waswashed with dichloromethane and carefully treated with 65% TFA/CH₂ Cl₂/0.5 M PhSH to remove the N-Boc group. The column was then carefullywashed with dichloromethane (20 ml) and DMF (20 ml) and then treatedwith the HOBt ester of Boc-γ prepared in situ (1 mmol, 200 μl DIEA, 1 mlDMF) and allowed to react for 1 hour. The CPG was washed with DMF (20ml), dichloromethane (20 ml), and dried in vacuo. The resin was removedfrom the column and placed in 1 ml 0.1 M NaOH at 55° C. for 15 hours.The CPG was removed by filtration, 1 ml of 1 M pH 7 triethylammoniumacetate was added and the mixture was purified by FPLC. The appropriatefractions were collected and concentrated in vacuo to giveBoc-γ-ImPyPy-CONH(CH₂)₁₂ P(O)₄ -TTTTTT^(m) C^(m) CTTT-3' (64) (SEQ IDNO:9) (343 nmole, 5% yield).

Characterization of Boc-γ-ImPyPy-CONH(CH₂)₁₂ P(O)₄ -TTTTTT^(m) C^(m)CTTT-3' (64). UV(H₂ O/DMSO)λ_(max) (ε) 260 (120,000), 304 (34,000);Analytic HPLC (5 nmole), r.t., 23.0 min., 6,000 u, Enzymatic digestion(5 nmole), 7.7 min. (mC, 779 u), 8.9 min. (T, 5873 u) 42.1 min.(peptide, 767 u (260), 597 u 340) MALDI-TOF MS, calc. M-1 4094.7, found4096.8.

Preparation of H₂ N-γ-ImPyPy-CONH(CH₂)₁₂ P(O)₄ -TTTTTT^(m) C^(m) CTTT-3'(65) (SEQ ID NO:10). Boc-γ-ImPyPy-CONH(CH₂)₁₂ P(O)₄ -TTTTTT^(m) C^(m)CTTT-3' (64) (330 nmole) was treated with 400 μl of 65% TFA/CH₂ Cl₂ /0.5M PhSH for 30 minutes. 2 ml of IM pH 7 triethylammonium acetate and 5 mlof water was added and the reaction mixture was vortexed, frozen, andlyophilized. The reaction mixture was then dissolved in 5 ml of 100 mMtriethylammonium acetate and purified by FPLC, appropriate fractionswere collected and concentrated in vacuo to give H₂N-γ-ImPyPy-CONH(CH₂)₁₂ P(O)₄ -TTTTTT^(m) C^(m) CTTT-3' (65) (240 nmole,70% yield). MALDI-TOF MS, calc. M-1 3995.5, found 3999.7

Preparation of EDTA-γ-ImPyPy-CONH(CH₂)₁₂ P(O)₄ -TTTTTT^(m) C^(m) CTTT-3'(66) (SEQ ID NO:11). H₂ N-γ-ImPyPy-CONH(CH₂)₁₂ P(O)₄ -TTTTTT^(m) C^(m)CTTT-3' (65) (SEQ ID NO:10) was dissolved in 500 μl 500 mM carbonatebuffer (pH 9.5). 10 mg of the monoanhydride of EDTA was added and thereaction allowed to proceed for 15 minutes. After 15 minutes 1 ml oftriethylammonium acetate, pH 7.0 was added with 4 ml of water and thereaction immediately purified by FPLC. The appropriate fractions werecollected and concentrated in vacuo to give EDTA-γ-ImPyPy-CONH(CH₂)₁₂P(O)₄ -TTTTTT^(m) C^(m) CTTT-3' (66) (70 nmole, 41% yield).

EXAMPLE 15

Automated Synthesis of Polyamides

The manual solid phase method for synthesis of pyrrole and imidazolepolyamides was adapted for use on an ABI 430A peptide synthesizer.Machine-assisted synthesis was performed on a 0.18 mmol scale (900 mgresin at 0.2 mmol/gram). Each cycle of amino acid addition involved:deprotection with approximately 80% TFA and 0.4 M thiophenol indichloromethane for 3 minutes, draining the reaction vessel, and thendeprotection for 17 minutes; 2 dichloromethane flow washes; an NMP flowwash; draining the reaction vessel; coupling for 1 hour with in situneutralization, addition of DMSO/NMP, coupling for 30 minutes, additionof DIEA, coupling for 30 minutes; draining the reaction vessel; washingwith dichloromethane, taking a resin sample for evaluation of theprogress of the synthesis by HPLC analysis; capping with aceticanhydride/DIEA in dichloromethane for 6 minutes; and washing withdichloromethane.

The synthesizer was left in the standard hardware configuration forNMP-HOBt protocols. Reagent positions 1 and 7 (FIG. 21) were DIEA,reagent position 2 was TFA/0.5 M thiophenol, reagent position 3 was 70%ethanolamine/methanol, reagent position 4 was acetic anhydride, reagentposition 5 was DMSO/NMP, reagent position 6 was methanol and reagentposition 8 was 0.48 M HBTU. All pyrrole and imidazole monomers werepreactivated, predissolved and filtered through an ISOLAB filter (cat.#QS-Q) before placing in a synthesis cartridge. Boc-Py-OBt ester (357mg, 1 mmol) was dissolved in 2 ml of DMF and filtered into a synthesiscartridge. Boc-imidazole monomer (125 mg, 0.5 mmol) and HOBT (135 mg,1.0 mmol) were dissolved in 500 μl of DMF, DCC (102 mg, 0.5 mmol) wasadded, and the mixture allowed to stand for 15 minutes. DMF (1.5 ml) wasthen added, DCU removed by filtration and the activated monomer placedin a synthesis cartridge. Boc-γ-Im-COOH or Boc-PyIm-COOH (100 mg,approximately 0.3 mmol) and HBTU (118 mg, 0.3 mmol) were dissolved in500 μl DMF, 100 μl DIEA was added and the solution allowed to stand for3 minutes. 1.5 ml of DMF was added and the solution filtered into asynthesis cartridge. For capping with imidazole-2-carboxylic acid, noactivation conditions were compatible with the delivery-line filters.Im-COOH (800 mg, approximately 6 mmol) and HBTU (1 g, 3 mmol) werecombined in 2.5 ml DMF, 1 ml DIEA was added and the mixture allowed tostand for 15 minutes. At the initiation of the coupling cycle, thesynthesis was interrupted, the reaction vessel vented with toggleswitches 0 and 2, the activated monomer filtered through a 0.2μ nylonfilter and added directly to the reaction vessel via syringe. Temporaryattachment of the resin sampling tube to a syringe, provides an easymethod for direct manual addition of regents to the reaction vessel. Forcoupling pyrrole to imidazole, Boc-Py-COOH (514 mg, 2 mmol) wasdissolved in 2 ml dichloromethane, DCC (420 mg, 2 mmol) was added, andthe solution allowed to stand for 10 minutes. DMAP (101 mg, 1 mmol) wasthen added and the solution allowed to stand for an additional 1 minute.The solution was filtered and manually added to the reaction vessel atthe initiation of coupling via syringe. For both coupling procedureswhere manual addition was necessary, the standard pyrrole-imidazolepolyamide activator cycle was used in conjunction with an emptysynthesis cartridge. Aliphatic amino acids (2 mmol) in 2 ml DMF wereactivated with HBTU (718 mg, 1.9 mmol), filtered and placed in asynthesis cartridge. Alternatively, the amino acid (1.5 mmol) was placeddry in a cartridge and 0.48 M HBTU (3 ml, 1.4 mmol) added using acalibrated delivery loop from reagent bottle eight, followed by theaddition of 1 ml DIEA from reagent bottle 7 using a calibrated deliveryloop, 3 minute mixing of the cartridge, direct transfer to theconcentrator without rinse, and subsequent transfer to the reactionvessel without rinse.

Preparation of ImPyPy-β-PyPyPy-G-Dp. The Polyamide ImPyPy-β-PyPyPy-G-Dpwas prepared by the general automated solid phase methods describedabove to yield a white powder. (17.2 mg, 57% recovery). HPLC, r.t. 26.5;UV(H₂ O/DMSO)λmax (ε), 246 (46,500), 312 (54,800); ¹ H NMR (DMSO-d₆) δ10.54 (s, 1H), 9.92 (s, 1H), 9.90 (m, 3H), 9.23 (br s, 1H), 8.27 (t, 1H,J=5.5 Hz), 8.06 (t, 1H, J=6.3 Hz), 8.03 (t, 1H, J=6.2 Hz), 7.39 (s, 1H),7.26 (d, 1H, J=1.7 Hz), 7.20 (m, 2H), 7.17 (m, 2H), 7.13 (m, 2H), 7.04(d, 1H), J=1.5 Hz), 6.87 (d, 1H, J=1.8 Hz), 6.83 (d, 1H, J=1.8 Hz), 3.97(s, 3H), 3.82 (m, 15H), 3.78 (d, 2H, J=3.4 Hz), 3.27 (m, 4H), 3.15 (m,2H), 3.79 (m, 2H), 2.76 (d, 6H, J=4.9 Hz), 1.78 (quintet, 2H, J=6.6 Hz);MALDI-TOF MS 950.2; FABMS m/e 949.458 (M⁺ H⁺ 949.455, calc. for C₄₅ H₅₇N₁₆ O₈).

                  TABLE 1                                                         ______________________________________                                        Polyamides synthesized by the solid phase method of this                      ______________________________________                                        invention.                                                                    ImPyPy-G-PyPyPy-G-Dp                                                          AcImPyPy-γ-PyPyPy-G-Ta                                                  AcImPyPy-γ-PyPyPy-G-Ta-EDTA                                             AcPyPyPy-γ-ImImPy-G-Dp                                                  ImPyPy-γ-PyPyPy-β-Dp                                               ImPyPy-β-PyPyPy-γ-ImPyPy-β-PyPyPy-β-Dp                   AcImPyPy-γ-PyPyPy-β-Dp                                             H.sub.2 N-γ-ImPyPy-β-PyPyPy-G-Dp                                   HOOC-Suc-ImPyPy-γ-PyPyPy-G-Dp                                           AcPyImPy-G-Dp                                                                 H.sub.2 N-PyPyPy-G-Dp                                                         ImPyPy-Dala-PyPyPy-G-Dp                                                       ImPyPy-γ-PyPyPy-G-Dp                                                    ImPyPy-Lala-PyPyPy-G-Dp                                                       ImPyPy-AIB-PyPyPy-G-Dp                                                        ImPyImPy-β-Dp                                                            ImPyPy-β-PyPyPy-G-Dp                                                     ImImPy-γ-PyPyPy-β-Dp                                               AcImPyPy-G-PyPyPy-G-Dp                                                        ImPyPy-G-PyPyPy-β-Dp                                                     ImPyPy-β-PyPyPy-γ-ImPyPy-β-PyPyPy-β-Ta                   ImPyPy-γ-ImPyPy-β-PyPyPy-G-Dp                                      ImPyPy-β-PyPyPy-G-Ta                                                     ImPyPy-G-PyPyPy-G-Ta                                                          AcPyPyPyPyPyPy-G-Ta                                                           AcImPyPy-γ-PyPyPy-Dp                                                    ImPyPyPyPyPyPy-G-Ta-EDTA                                                      ImPyPy-Lglu-PyPyPy-G-Dp                                                       ImPyPyPyPyPyPy-G-Dp                                                           ImPyPyPyPyPyPy-G-ED.                                                          AcImPyPy-G-PyPyPy-G-Ta-EDTA                                                   AcImImPy-γ-PyPyPy-G-Ta                                                  AcPyPyPy-γ-ImImPy-G-Ta                                                  AcPyPyPy-γ-ImImPy-β-Dp                                             AcPyPyPy-γ-ImImPy-G-Dp                                                  H.sub.2 N-ImPyPy-G-PyPyPy-G-Dp                                                EDTA-γ-ImPyPy-β-PyPyPy-G-Dp                                        ImPyPy-γ-ImPyPy-G-PyPyPy-G-Dp                                           H.sub.2 N-γ-ImPyPy-β-PyPyPy-G-Ta                                   AcImImPy-γ-PyPyPy-G-Dp                                                  AcImPyPy-γ-PyPyPy-G-Dp                                                  ImPy-G-Py-γ-ImPy-G-Py-β-Dp                                         ImImPy-γ-ImPyPy-β-PyPyPy-G-Dp                                      ImPyImPy-γ-ImPyImPy-β-Dp                                           ImPyImPy-γ-PyPyPyPy-β-Dp                                           ImImPyPy-γ-PyPyPyPy-β-Dp                                           ImPyPy-β-PyPyPy-G-Ta-EDTA                                                ImPyPy-G-PyPyPy-G-Ta-EDTA                                                     AcImImPy-γ-PyPyPy-β-Dp                                             AcImPyPy-G-PyPyPy-G-Ta                                                        ImPyPy-G-PyPyPy-G-ED.                                                         ImPyPy-γ-ImPyPy-β-Dp                                               AcImPyPyPyPyPyPy-G-Ta-EDTA                                                    AcPyPyPy-γ-ImImPy-G-Ta-EDTA                                             ImPyPy-transcyclopropyl-PyPyPy-β-Dp                                      AcImPyPy-G-PyPyPy-G-Ta                                                        PyPyPy-γ-ImImPy-G-Dp                                                    ImImIm-β-PyPyPy-β-Dp                                                AcPyPyImPy-γ-PyPyPyPy-β-Dp                                         AcImImPy-γ-PyPyPy-G-Dp                                                  H.sub.2 N-β-PyPyPy-γ-ImImPy-β-β-β-β-PyPyPy-    γ-ImImPy-β-Dp                                                      (automated synthesis)                                                         ImPyPyPy-γ-ImPyPyPy-β-Dp                                           PyPyPy-γ-ImImPy-β-Dp                                               PyPyPy-γ-ImImPy-G-Dp                                                    DM-γ-ImPyPy-β-PyPyPy-β-Dp                                     ImPyPy-β-ImImPyPy-γ-ImImPyPy-β-Dp                             ImPyPy-β-PyPyPy-β-Dp                                                ImImPyPy-γ-ImImPyPy-β-Dp                                           ImPyPy-γ-β-β-β-β-Dp                                 ImPyPy-γ-β-PyPy-β-Dp                                          ImPyPy-γ-ImPyPy-β-PyPyPy-G-Ta-EDTA                                 H.sub.2 N-γ-ImPyPyPy-γ-PyPyPyPy(G-Dp)-COOH                        ImImImIm-γ-PyPyPyPy-β-Dp                                           ImPyPyPy-β-ImImPyPy-γ-ImImPyPy-β-Ta-EDTA                      ImPyPyPy-γ-PyPyPyPy-β-Dp                                           H.sub.2 N-ε-ImPyPy-G-PyPyPy-G-Dp                                      DMγ-ImPyPy-γ-ImPyPy-β-ED                                     ImPyPyPy-γ-PyPyPyPy-Ta                                                  ImPyPyPy-γ-PyPyPyPy-Ta-EDTA                                             ImPyPyPy-γ-ImPyPyPy-β-Ta                                           ImPyPyPy-γ-ImPyPyPy-β-Ta-EDTA                                      ImPyPyPy-γ-ImImImPy-β-Ta                                           ImPyPyPy-γ-ImImImPy-β-PyPyPyPy-β-Ta                           ImPyPy-Dala-PyPyPy-β-Dp                                                  ImPyPy-Lala-PyPyPy-β-Dp                                                  ImPyPy-β-PyPyPy-Dala-Dp                                                  ImPyPy-β-PyPyPy-Lala-Dp                                                  ImPyPy-γ-.sup.m PyPyPy-β-Dp                                        ImPy.sup.m Py-γ-PyPyPy-β-Dp                                        ImPyPy-β-Py.sup.m PyPy-β-Dp                                         Im.sup.m PyPy-β-PyPyPy-β-Dp                                         EDTA-γ-ImPyPy-G-PyPyPy-G-Dp                                             EDTA-γ-ImPyPy-G-PyPyPy-G-Ta-EDTA                                        EDTA-γ-ImPyPy-β-PyPyPy-G-Ta-EDTA                                   ______________________________________                                         *All compounds listed have be characterized by HPLC, .sup.1 HNMR, MALDITO     mass spectroscopy and in some cases .sup.13 C NMR.                            Abbreviations.                                                                Im = Imidazole                                                                Py = Pyrrole                                                                  G = Glycine                                                                   Dp = Dimethylaminopropylamine                                                 Ac = Acetyl                                                                   EDTAethylenediaminetetraacetic acid                                           ED = ethylenediamine                                                          Ta = 3,3diamino-N-methylpropylamine                                           Lala = Lalanine                                                               β = alanine                                                              γ = aminobutyric acid                                                   Dala = Dalanine                                                               AIB = alphaisobutyric acid                                                    DMγ = N,Ndimethyl-aminobutyric acid                                     Suc = Succinic acid                                                           Lglu = LGlutamic acid                                                         ε = aminohexanoic acid                                           

                  TABLE 2                                                         ______________________________________                                        Standard protocol for manual synthesis of                                     minor groove binding polyamides.                                              SYNTHESIS CYCLE                                                                            REAGENTS       TIME/MODE                                         ______________________________________                                        1) Deprotect 65% TFA/CH.sub.2 Cl.sub.2 /PhSH                                                              1 × 30 s flow                                                           1 × 1 min shake                                                         1 × 30 s flow                                                           1 × 20 min shake                            2) Wash      CH.sub.2 Cl.sub.2                                                                            1 × 1 min flow                                           DMF            1 × 30 s flow                                                           1 × 1 min shake                                        (take sample for picric acid test)                                 3) Couple    HOBt acid, DIEA                                                                              45 min shake                                                 (take sample for picric acid test)                                 4) Wash      DMF            1 × 30 s flow                                            CH.sub.2 Cl.sub.2                                                                            1 × 30 s flow                               ______________________________________                                    

                                      TABLE 3                                     __________________________________________________________________________    Apparent First-Order Association Constants (M.sup.-1) for selected            polyamides..sup.a                                                                         Polyamide                                                                     ImPyPy-G-                                                                             ImPyPy-G-                                                                             ImPyPy-G-                                                                             SEQ ID                                    Binding Site                                                                              PyPyPy-Dp                                                                             PyPyPy-G-Dp                                                                           PyPyPy-β-Dp                                                                      NO:                                       __________________________________________________________________________    5'-AAAAAGACAAAAA-3'                                                                       1.1 (±0.1) × 10.sup.8                                                        7.0 (±1.2) × 10.sup.7                                                        1.0 (±0.2) × 10.sup.8                                                        2                                         5'-ATATAGACATATA-3'                                                                       6.6 (±0.4) × 10.sup.6                                                        ≈3.5 (±1.4) × 10.sup.6                                               1.0 (±0.2) × 10.sup.7                                                        3                                         5'-TGTTAAACA-3'                                                                           1.4 (±0.1) × 10.sup.8                                                        ≈1.7 (±0.7) × 10.sup.6                                               3.4 (±0.5) × 10.sup.7                                                        4                                         5'-TGTAAAACG-3                                                                            5.5 (±0.3) × 10.sup.7                                                        ≈3.0 (±1.5) × 10.sup.6                                               4.0 (±1.3) × 10.sup.7                                                        ?                                         5'-TGTGCTGCAAG-3'                                                                         5.4 (±0.2) × 10.sup.7                                                        <1 × 10.sup.6                                                                   3.7 (±1.0) × 10.sup.7                                                        6                                         __________________________________________________________________________     .sup.a Values reported are the mean values obtained from three DNase I        footprint titration experiments. The standard deviation for each value is     indicated in parentheses.                                                

                  TABLE 4*                                                        ______________________________________                                        Illustrative Cyclic Polyamides.                                               ______________________________________                                        cyclo-(ImPyImPy-γ-ImPyImPy-(G-Dp)-γ-)                             cyclo(ImPyImPy-γ-ImPyImPy(G-Dp))                                        cyclo(ImPyPyPy-γ-PyPyPyPy(G-Dp))                                        H.sub.2 N-γ-ImPyImPy-γ-ImPyImPy(G-Dp)-COOH                        H.sub.2 N-γ-ImPyImPy-γ-ImPyImPy(G-Dp)-COOH                        ______________________________________                                         *Abbreviations.                                                               Im = Imidazole                                                                Py = Pyrrole                                                                  G = Glycine                                                                   Dp = Dimethylaminopropylamine                                                 γ = aminobutyric acid                                              

                  TABLE 5                                                         ______________________________________                                        Oligonucleotide-polyamide conjugates.                                                                       SEQ                                                                           ID                                              OLIGONUCLEOTIDE-POLYAMIDE CONJUGATE*                                                                        NO:                                             ______________________________________                                        ImPyPy-C.sub.6 -P(O).sub.4 TTTTTT.sup.m C.sup.m CTTT-3'                                                      7                                              AcImPyPy-C.sub.12 -P(O).sub.4 TTTTTT.sup.m C.sup.m CTTT-3'                                                   8                                              Boc-γ-ImPyPy-C.sub.12 -P(O).sub.4 TTTTTT.sup.m C.sup.m CTTT-3'                                         9                                              H.sub.2 N-γ-ImPyPy-C.sub.12 -P(O).sub.4 TTTTTT.sup.m C.sup.m            CTTT-3'                       10                                              EDTA-γ-ImPyPy-C.sub.12 -P(O).sub.4 TTTTTT.sup.m C.sup.m CTTT-3'                                       11                                              ImPyPy-C.sub.6 -P(O).sub.4 TTT.sup.m C.sup.m CTTTTTT-3'                                                     12                                              AcImPyPy-C.sub.12 -P(O).sub.4 -TTT.sup.m C.sup.m CTTTTTT-3'                                                 13                                              H.sub.2 N-γ-ImPyPy-C.sub.6 -P(O).sub.4 -TTT.sup.m C.sup.m CTTTTTT-3'                                  14                                              H.sub.2 N-γ-ImPyPy-C.sub.8 -P(O).sub.4 -TTT.sup.m C.sup.m CTTTTTT-3'                                  15                                              H.sub.2 N-γ-ImPyPy-C.sub.10 -P(O).sub.4 -TTT.sup.m C.sup.m CTTTTTT-3    '                             16                                              H.sub.2 N-γ-ImPyPy-C.sub.12 -P(O).sub.4 -TTT.sup.m C.sup.m CTTTTTT-3    '                             17                                              H.sub.2 N-γ-ImPyPy-C.sub.6 -P(O).sub.4 (CH.sub.2).sub.8 (NH)TTT.sup.    m C.sup.m CTTTTTT-3'          18                                              Dp-G-PyPyPy-G-PyPyIm-ε-DSA-(NH)TTT.sup.m C.sup.m CTTTTTT-3'                                         19                                              Dp-G-PyPyPy-G-PyPyIm-ε-DSG-(NH)TTT.sup.m C.sup.m CTTTTTT-3'                                         20                                              Dp-G-PyPyPy-G-PyPyIm-ε-DSS-(NH)TTT.sup.m C.sup.m CTTTTTT-3'                                         21                                              DM-γ-ImPyPy-γ-ImPyPy-β-ED-DSG-(NH)TTT.sup.m C.sup.m          CTTTTTT-3'                    22                                              DMγ-ImPyPy-γ-ImPyPy-β-ED-DSA-(NH)TTT.sup.m C.sup.m           CTTTTTT-3'                    23                                              ______________________________________                                         *Abbreviations.                                                               C.sub.6 represents CONH(CH.sub.2).sub.6                                       C.sub.12 represents CONH(CH.sub.2).sub.12                                     .sup.m C = 5methylcytidine                                                    ETDA = ethylenediaminetetraacetic acid                                        γ = aminobutyric acid                                                   DSA = Adipic acid                                                             DSG = Glutaric acid                                                           DSS = Suberic acid                                                            (NH)T = 2',5dideoxy-5aminothymidine                                           Im = Imidazole                                                                Py = Pyrrole                                                                  G = Glycine                                                                   Dp = Dimethylaminopropylamine                                                 ε = aminohexanoic acid                                                β = alanine                                                              ED = ethylenediamine                                                          C.sub.8 = CONH(CH.sub.2).sub.8                                                C.sub.10 = CONH(CH.sub.2).sub.10                                         

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 23                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 12 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 #       12                                                                    - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 13 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 #      13                                                                     - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 13 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 #      13                                                                     - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 9 nucleotid - #es                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 #          9                                                                  - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 12 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 #       12                                                                    - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 11 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 #       11                                                                    - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 12 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: N                                                               (B) LOCATION: 1                                                     #N is ImPyPy-CONH(CH2)6-P(O)4ON:                                              -     (ix) FEATURE:                                                           #Base     (A) NAME/KEY: Modified                                                        (B) LOCATION: 8-9                                                   #N is 5'-methylcytidineORMATION:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 #       12                                                                    - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 12 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: N                                                               (B) LOCATION: 1                                                     #N is AcImPyPy-CONH(CH2)12P(O)4:                                              -     (ix) FEATURE:                                                           #Base     (A) NAME/KEY: Modified                                                        (B) LOCATION: 8-9                                                   # N is 5'-methylcytidineRMATION:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 #       12                                                                    - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 12 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: N                                                               (B) LOCATION: 1                                                     ImPyPy-CONH(CH2)12P(O)4ORMATION:                                              -     (ix) FEATURE:                                                           #Base     (A) NAME/KEY: Modified                                                        (B) LOCATION: 8-9                                                   # N is 5'-methylcytidineRMATION:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 #       12                                                                    - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 12 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: N                                                               (B) LOCATION: 1                                                     ImPyPy-CONH(CH2)12P(O)4ORMATION:                                              -     (ix) FEATURE:                                                           #Base     (A) NAME/KEY: Modified                                                        (B) LOCATION: 8-9                                                   # N is 5'-methylcytidineRMATION:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                #       12                                                                    - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 12 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: N                                                               (B) LOCATION: 1                                                     ImPyPy-CONH(CH2)12P(O)4ORMATION:                                              -     (ix) FEATURE:                                                           #Base     (A) NAME/KEY: Modified                                                        (B) LOCATION: 8-9                                                   # N is 5'-methylcytidineRMATION:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                #       12                                                                    - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 12 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: N                                                               (B) LOCATION: 1                                                     # N is ImPyPy-C6-P(O)4FORMATION:                                              -     (ix) FEATURE:                                                           #Base     (A) NAME/KEY: Modified                                                        (B) LOCATION: 5-6                                                   # N is 5'-methylcytidineRMATION:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                #       12                                                                    - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 12 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: N                                                               (B) LOCATION: 1                                                     # N is AcImPyPy-C12-P(O)4MATION:                                              -     (ix) FEATURE:                                                           #Base     (A) NAME/KEY: Modified                                                        (B) LOCATION: 5-6                                                   # N is 5'-methylcytidineRMATION:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                #       12                                                                    - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 12 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: N                                                               (B) LOCATION: 1                                                     ImPyPy-C6-P(O)4THER INFORMATION:                                              -     (ix) FEATURE:                                                           #Base     (A) NAME/KEY: Modified                                                        (B) LOCATION: 5-6                                                   # N is 5'-methylcytidineRMATION:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                #       12                                                                    - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 12 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: N                                                               (B) LOCATION: 1                                                     ImPyPy-C8-P(O)4THER INFORMATION:                                              -     (ix) FEATURE:                                                           #Base     (A) NAME/KEY: Modified                                                        (B) LOCATION: 5-6                                                   # N is 5'-methylcytidineRMATION:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                #       12                                                                    - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 12 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: N                                                               (B) LOCATION: 1                                                     ImPyPy-C10-P(O)4HER INFORMATION:                                              -     (ix) FEATURE:                                                           #Base     (A) NAME/KEY: Modified                                                        (B) LOCATION: 5-6                                                   # N is 5'-methylcytidineRMATION:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                #       12                                                                    - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 12 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: N                                                               (B) LOCATION: 1                                                     ImPyPy-C12-P(O)4HER INFORMATION:                                              -     (ix) FEATURE:                                                           #Base     (A) NAME/KEY: Modified                                                        (B) LOCATION: 5-6                                                   # N is 5'-methylcytidineRMATION:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                #       12                                                                    - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 12 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: N                                                               (B) LOCATION: 1                                                     ImPyPy-C6-P(O)4(CH2)8(NH)MATION:                                              -     (ix) FEATURE:                                                           #Base     (A) NAME/KEY: Modified                                                        (B) LOCATION: 5-6                                                   # N is 5'-methylcytidineRMATION:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                #       12                                                                    - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 12 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: N                                                               (B) LOCATION: 1                                                     # N is GpyPyImnDSA(NH)FORMATION:                                              -     (ix) FEATURE:                                                           #Base     (A) NAME/KEY: Modified                                                        (B) LOCATION: 5-6                                                   # N is 5'-methylcytidineRMATION:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                #       12                                                                    - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 12 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: N                                                               (B) LOCATION: 1                                                     # N is Dp-G-PyPyPy-G-PyPyIm-n-DSG-                                                      (NH)                                                                -     (ix) FEATURE:                                                           #Base     (A) NAME/KEY: Modified                                                        (B) LOCATION: 5-6                                                   # N is 5'-methylcytidineRMATION:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                #       12                                                                    - (2) INFORMATION FOR SEQ ID NO:21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 12 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: N                                                               (B) LOCATION: 1                                                     # N is Dp-G-PyPyPy-G-PyPyIm-n-DSS-                                                      (NH)                                                                -     (ix) FEATURE:                                                           #Base     (A) NAME/KEY: Modified                                                        (B) LOCATION: 5-6                                                   # N is 5'-methylcytidineRMATION:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                #       12                                                                    - (2) INFORMATION FOR SEQ ID NO:22:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 12 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: N                                                               (B) LOCATION: 1                                                     ImPyPy-a-ED-DSG-HER INFORMATION:                                                        (NH)                                                                -     (ix) FEATURE:                                                           #Base     (A) NAME/KEY: Modified                                                        (B) LOCATION: 5-6                                                   #N is 5'-methylcytidineORMATION:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                #       12                                                                    - (2) INFORMATION FOR SEQ ID NO:23:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 12 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: N                                                               (B) LOCATION: 1                                                     ImPyPy-a-ED-DSA-HER INFORMATION:                                                        (NH)                                                                -     (ix) FEATURE:                                                           #Base     (A) NAME/KEY: Modified                                                        (B) LOCATION: 5-6                                                   # N is 5'-methylcytidineRMATION:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                #       12                                                                    __________________________________________________________________________

What is claimed is:
 1. A method for preparing a polyamide containingimidazole and pyrrole carboxamide groups on a solid supportcomprising:(a) preparing a resin for attachment of the polyamide; (b)reacting an amino acid with reagents to provide an amino acid containingan amino group which is protected and a carboxyl reactive with an aminofunctionality. (c) sequentially deprotecting the amino acid and addingthe protected and reactive amino acids to the solid support beginningwith the carboxy terminal amino acid, thereby forming the desiredpolyamide; (d) cleaving the polyamide from the resin; and (e) purifyingthe polyamide.
 2. The method of claim 1 further comprising:(f) reactingthe polyamide with EDTA prior to purification.
 3. The method of claim 1wherein the resin is a polystyrene resin.
 4. The method of claim 1wherein said polyamide is attached to said resin through a resin linkageagent selected from the group consisting oftert-butyloxycarbonylaminoacyl-pyrrole-4-(oxymethyl)phenylacetic acid(Boc-Py-PAM),tert-butyloxycarbonylaminoacyl-pyrrole-4-(oxymethyl)benzoic acid(Boc-Py-BAM),tert-butyloxycarbonylaminoacyl-imidazole4-(oxymethyl)phenylacetic acid(Boc-Im-PAM),tert-butyloxycarbonylaminoacyl-pyrrole4-(oxymethyl)phenylaceticacid-G-4-(oxymethyl)phenylacetic acid (Boc-Py-PAM-G-PAM),tert-butyloxycarbonylaminoacyl-pyrrole-4(oxymethyl)benzoicacid-G4-(oxymethyl)phenylacetic acid (Boc-Py-BAM-G-PAM),tert-butyloxycarbonylaminoacyl-pyrrole-G4-(oxymethyl)phenylacetic acid(Boc-Py-G-PAM),tert-butyloxycarbonylaminoacyl-pyrrole-β-4-(oxymethyl)phenylacetic acid(Boc-Py-β-PAM),tert-butyloxycarbonylaminoacyl-imidazole-G4-(oxymethyl)phenylacetic acid(Boc-Im-G-PAM),tert-butyloxycarbonylaminoacyl-imidazole-β-4-(oxymethyl)phenylaceticacid (Boc-Im-β-PAM) andtert-butyloxycarbonylaminoacyl-imidazole-4-(oxymethyl)benzoic acid(Boc-Im-BAM).
 5. A method for preparing a polyamide containing N-methyl4-imidazolecarboxamide and N-methyl-pyrrolecarboxamide comprising:(a)sequentially adding amino acids with an amino group protected withtert-butoxycarbonyl or 9-fluorenylmethylcarbonyl and a carboxy groupthat has been reacted to form an activated ester or a symmetricanhydride to an amino substituted solid support, beginning with thecarboxy terminal amino acid, thereby forming a polyamide having aprotected amino acid; (b) deprotecting the amino terminal protectedamino acid of the polyamide; and (c) removing the polyamide from thesolid support.
 6. The method of claim 1 wherein said polyamide is bondedto said resin through a spacer selected from the group consisting ofglycine, β-alanine (β), glycine-PAM, and glycine-BAM.
 7. The method ofclaim 6 wherein the substitution ratio is 0.2 to 0.3 mmol/gram.
 8. Themethod of claim 1 wherein said amino acid is an amino acid monomer or anamino acid dimer.
 9. The method of claim 8 wherein the amino acidmonomer is selected from the group consisting of a pyrrole amino acid,an imidazole amino acid, an aromatic amino acid and an aliphatic aminoacid.
 10. The method of claim 9 wherein said pyrrole amino acid has thefollowing structure: ##STR28## wherein R₁ is selected from the groupconsisting of H, CH₃, OH, NH₂, Cl and CF₃ ; and R₂ is selected from thegroup consisting of a C1-C10 alkyl group, a C1-C10 alkene, and a C1-C10alkyne.
 11. The method of claim 9 wherein said imidazole amino acid hasthe following structure: ##STR29## wherein R₂ is selected from the groupconsisting of a C1-C10 alkyl group, a C1-C10 alkene, and a C1-C10alkyne.
 12. The method of claim 8 wherein said amino acid dimer has thefollowing structure: ##STR30## wherein R₂ is selected from the groupconsisting of a C1-C10 alkyl group, a C1-C10 alkene, and a C1-C 10alkyne and R₃ is an amino acid selected from the group consisting of apyrrole amino acid, an imidazole amino acid, an aromatic amino acid andan aliphatic amino acid.
 13. The method of claim 1 wherein the aminogroup of the amino acid is protected with a protecting group selectedfrom the group consisting of tert-butoxy carbonyl (Boc-) and9-fluoroenylmethylcarbonyl (Fmoc-).
 14. The method of claim 1 whereinthe carboxyl of the amino acid is the oxybenzotriazole (-OBt) ester. 15.The method of claim 1 wherein the carboxyl of the amino acid is thesymmetric anhydride.
 16. The method of claim 1 wherein the amino acidsof the polyamide are deprotected by reaction with trifluoroacetic acid.17. The method of claim 16 wherein a cation scavenger is present and isselected from the group consisting of thiophenol, methyl ethyl sulfideand ethanedithiol.
 18. The method of claim 1 wherein the polyamide iscleaved from the resin with Pd(OAc)₂.
 19. The method of claim l whereinthe polyamide is cleaved from the resin by aminolysis with an amineselected from the group consisting of dimethylaminopropylamine (Dp),3,3'-diamino-Nmethyldipropylamine (Ta), or ethylene-diamine (ED), andβ-alanine(β).
 20. A polyamide produced according to the method of claim1 containing imidazole and pyrrole carboxamide groups wherein saidpolyamide contains an aliphatic amino acid selected from the groupconsisting of glycine, alanine and γ-aminobutyric acid and wherein saidpolyamide contains at least seven monomer units.
 21. The polyamide ofclaim 20 wherein said polyamide is selected from the group consistingof:ImPyPy-G-PyPyPy-G-Dp, AcImPyPy-γ-PyPyPy-G-Ta,AcImPyPy-γ-PyPyPy-G-Ta-EDTA, AcPyPyPy-γ-ImImPy-G-Dp,ImPyPy-γ-PyPyPy-β-Dp, ImPyPy-β-PyPyPy-γ-ImPyPy-β-PyPyPy-β-Dp,AcImPyPy-γ-PyPyPy-β-Dp, H₂ N-γ-ImPyPy-β-PyPyPy-G-Dp,HOOC-Suc-ImPyPy-γ-PyPyPy-G-Dp, AcPyImPy-G-Dp, H₂ N-PyPyPy-G-Dp,ImPyPy-Dala-PyPyPy-G-Dp, ImPyPy-γ-PyPyPy-G-Dp, ImPyPy-Lala-PyPyPy-G-Dp,ImPyPy-AIB-PyPyPy-G-Dp, ImPyImPy-β-Dp, ImPyPy-β-PyPyPy-G-Dp,ImImPy-γ-PyPyPy-β-Dp, AcImPyPy-G-PyPyPy-G-Dp, ImPyPy-G-PyPyPy-β-Dp,ImPyPy-β-PyPyPy-γ-ImPyPy-β-PyPyPy-β-Ta, ImPyPy-γ-ImPyPy-β-PyPyPy-G-Dp,ImPyPy-β-PyPyPy-G-Ta, ImPyPy-G-PyPyPy-G-Ta, AcPyPyPyPyPyPy-G-Ta,AcImPyPy-γ-PyPyPy-Dp, ImPyPyPyPyPyPy-G-Ta-EDTA, ImPyPy-Lglu-PyPyPy-G-Dp,ImPyPyPyPyPyPy-G-Dp, ImPyPyPyPyPyPy-G-ED, AcImPyPy-G-PyPyPy-G-Ta-EDTA,AcImImPy-γ-PyPyPy-G-Ta, AcPyPyPy-γ-ImImPy-G-Ta, AcPyPyPy-γ-ImImPy-β-Dp,AcPyPyPy-γ-ImImPy-G-Dp, H₂ N-ImPyPy-G-PyPyPy-G-Dp,EDTA-γ-ImPyPy-β-PyPyPy-G-Dp, ImPyPy-γ-ImPyPy-G-PyPyPy-G-Dp, H₂N-γ-ImPyPy-β-PyPyPy-G-Ta, AcImImPy-γ-PyPyPy-G-Dp,AcImPyPy-γ-PyPyPy-G-Dp, ImPy-G-Py-γ-ImPy-G-Py-β-Dp,ImImPy-γ-ImPyPy-β-PyPyPy-G-Dp, ImPyImPy-γ-ImPyImPy-β-Dp,ImPyImPy-γ-PyPyPyPy-β-Dp, ImImPyPy-γ-PyPyPyPy-β-Dp,ImPyPy-β-PyPyPy-G-Ta-EDTA, ImPyPy-G-PyPyPy-G-Ta-EDTA,AcImImPy-γ-PyPyPy-β-Dp, AcImPyPy-G-PyPyPy-G-Ta, ImPyPy-G-PyPyPy-G-ED,ImPyPy-γ-ImPyPy-β-Dp, AcImPyPyPyPyPyPyPy-G-Ta-EDTA,AcPyPyPy-γ-ImImPy-G-Ta-EDTA, ImPyPy-transcyclopropyl-PyPyPy-β-Dp,AcImPyPy-G-PyPyPy-G-Ta, PyPyPy-γ-ImImPy-G-Dp, ImImIm-β-PyPyPy-β-Dp,AcPyPyImPy-γ-PyPyPyPy-β-Dp, AcImImPy-γ-PyPyPy-G-Dp, H₂N-β-PyPyPy-γ-ImImPy-β-β-β-β-PyPyPy-γ-ImImPy-β-Dp,ImPyPyPy-γ-ImPyPyPy-β-Dp, PyPyPy-γ-ImImPy-β-Dp, PyPyPy-γ-ImImPy-G-Dp,DM-γ-ImPyPy-β-PyPyPy-β-Dp, ImPyPy-β-ImImPyPy-γ-ImImPyPy-β-Dp,ImPyPy-β-PyPyPy-β-Dp, ImImPyPy-γ-ImImPyPy-β-Dp, ImPyPy-γ-β-β-β-β-Dp,ImPyPy-γ-β-PyPy-β-Dp, ImPyPy-γ-ImPyPy-β-PyPyPy-G-Ta-EDTA, H₂N-γ-ImPyPyPy-γ-PyPyPyPy(G-Dp)-COOH, ImImImIm-γ-PyPyPyPy-β-Dp,ImPyPyPy-β-ImImPyPy-γ-ImImPyPy-β-Ta-EDTA, ImPyPyPy-γ-PyPyPyPy-β-Dp, H₂N-ε-ImPyPy-G-PyPyPy-G-Dp, DMγ-ImPyPy-γ-ImPyPy-β-ED,ImPyPyPy-γ-PyPyPyPy-Ta, ImPyPyPy-γ-PyPyPyPy-Ta-EDTA,ImPyPyPy-γ-ImPyPyPy-β-Ta, ImPyPyPy-γ-ImPyPyPy-β-Ta-EDTA,ImPyPyPy-γ-ImImImPy-β-Ta, ImPyPyPy-γ-ImImImPy-β-PyPyPyPy-β-Ta,ImPyPy-Dala-PyPyPy-β-Dp, ImPyPy-Lala-PyPyPy-β-Dp,ImPyPy-β-PyPyPy-Dala-Dp, ImPyPy-β-PyPyPy-Lala-Dp, ImPyPy-γ-^(m)PyPyPy-β-Dp, ImPy^(m) Py-γ-PyPyPy-β-Dp, ImPyPy-β-Py^(m) PyPy-β-Dp,Im^(m) PyPy-β-PyPyPy-β-Dp, EDTA-γ-ImPyPy-G-PyPyPy-G-Dp,EDTA-γ-ImPyPy-G-PyPyPy-G-Ta-EDTA, and EDTA-γ-ImPyPy-β-PyPyPy-G-Ta-EDTA.22. A polyamide capable of forming hydrogen bonds with nucleotide basesin the minor-groove of double-stranded DNA produced by the method ofclaim I and wherein said polyamide contains at least seven monomerunits.
 23. A method for preparing a cyclic polyamide on a solid supportcomprising:(a) preparing a resin for attachment of the polyamide; (b)reacting the resin with a protected allyl ester pyrrole monomer havingthe following structure: ##STR31## (c) reacting an amino acid withreagents to provide an amino group which is protected and a carboxylreactive with an amino functionality; (d) sequentially adding theprotected and reactive amino acids to the solid support beginning withthe carboxy terminal amino acid, thereby forming the desired polyamide;(e) removing the allyl ester with a Pd catalyst; (f) cleaving thepolyamide from the resin; and (g) cyclizing and purifying the cyclizedpolyamide.
 24. A polyamide containing imidazole and pyrrole carboxamidegroups produced according to the method of claim 23 and wherein saidpolyamide contains at least four carboxamide pairs.
 25. The polyamide ofclaim 24 wherein said polyamide is selected from the group consistingof:cyclo-(ImPyImPy-γ-ImPyImPy-(G-Dp)-γ-),cyclo(ImPyImPy-γ-ImPyImPy(G-Dp)), cyclo(ImPyPyPy-γ-PyPyPyPy(G-Dp)), H₂N-γ-ImPyImPy-γ-ImPyImPy(G-Dp)-COOH, H₂N-γ-ImPyImPy-γ-ImPyImPy(G-Dp)-COOH.
 26. A cyclic polyamide capable offorming hydrogen bonds with nucleotide bases in the minor-groove ofdouble-stranded DNA produced by the method of claim 1 and wherein saidpolyamide contains at least four carboxamide pairs.
 27. A method forpreparing a polyamide containing N-methyl imidazolecarboxamide andN-methyl-pyrrolecarboxamide on a solid support comprising:(a) preparingan amino substituted polystyrene resin solid support for attachment ofthe polyamide; (b) protecting the amino group of an amino acid with amember of the group consisting of tert-butoxycarbonyl or9-fluorenylmethylcarbonyl and reacting the carboxyl group to form theoxybenztriazole ester or a symmetric anhydride; (c) sequentiallydeprotecting the amino acid and adding the protected amino acids to thesolid support beginning with the carboxy terminal amino acid, therebyforming the desired polyamide; (d) cleaving the polyamide from the resinby hydrogenolysis; and (e) purifying the polyamide.
 28. The method ofclaim 27 wherein said polyamide comprises an aliphatic amino acidselected from the group consisting of glycine, alanine andγ-aminobutyric acid.
 29. The polyamide of claim 20 wherein saidpolyamide is selected from the group consistingof:AcImPyPy-γ-PyPyPy-G-Ta-EDTA, ImPyPyPyPyPyPy-G-Ta-EDTA,AcImPyPy-G-PyPyPy-G-Ta-EDTA, EDTA-γ-ImPyPy-β-PyPyPy-G-Dp,ImPyPy-β-PyPyPy-G-Ta-EDTA, ImPyPy-G-PyPyPy-G-Ta-EDTA,AcImPyPyPyPyPyPy-G-Ta-EDTA, AcPyPyPy-γ-ImImPy-G-Ta-EDTA,ImPyPy-γ-ImPyPy-β-PyPyPy-G-Ta-EDTA,ImPyPyPy-β-ImImPyPy-γ-ImImPyPy-β-Ta-EDTA, ImPyPyPy-γ-PyPyPyPy-Ta-EDTA,ImPyPyPy-γ-ImPyPyPy-β-Ta-EDTA, EDTA-γ-ImPyPy-G-PyPyPy-G-Dp,EDTA-γ-ImPyPy-G-PyPyPy-G-Ta-EDTA, and EDTA-γ-ImPyPy-β-PyPyPy-G-Ta-EDTA.30. The polyamide of claim 20 wherein said polyamide is selected fromthe group consisting of:AcImPyPy-γ-PyPyPy-G-Ta,AcImPyPy-γ-PyPyPy-G-Ta-EDTA, AcPyPyPy-γ-ImImPy-G-Dp,ImPyPy-γ-PyPyPy-β-Dp, ImPyPy-β-PyPyPy-γ-ImPyPy-β-PyPyPy-β-Dp,AcImPyPy-γ-PyPyPy-β-Dp, H₂ N-γ-ImPyPy-β-PyPyPy-G-Dp,HOOC-Suc-ImPyPy-γ-PyPyPy-G-Dp, Im-PyPy-γ-PyPyPy-G-Dp,ImImPy-γ-PyPyPy-β-Dp, ImPyPy-β-PyPyPy-γ-ImPyPy-β-PyPyPy-β-Ta,ImPyPy-γ-ImPyPy-β-PyPyPy-G-Dp, AcImPyPy-γ-PyPyPy-Dp,AcImImPy-γ-PyPyPy-G-Ta, AcPyPyPy-γ-ImImPy-G-Ta, AcPyPyPy-γ-ImImPy-β-Dp,AcPyPyPy-γ-ImImPy-G-Dp, EDTA-γ-ImPyPy-β-PyPyPy-G-Dp,ImPyPy-γ-ImPyPy-G-PyPyPy-G-Dp, H₂ N-γ-ImPyPy-β-PyPyPy-G-Ta,AcImImPy-γ-PyPyPy-G-Dp, AcImPyPy-γ-PyPyPy-G-Dp,ImPy-G-Py-γ-ImPy-G-Py-β-Dp, ImImPy-γ-ImPyPy-β-PyPyPy-G-Dp,ImPyImPy-γ-ImPyImPy-β-Dp, ImPyImPy-γ-PyPyPyPy-β-Dp,ImImPyPy-γ-PyPyPyPy-β-Dp, AcImImPy-γ-PyPyPy-β-Dp, ImPyPy-γ-ImPyPy-β-Dp,AcPyPyPy-γ-ImImPy-G-Ta-EDTA, PyPyPy-γ-ImImPy-G-Dp,AcPyPyImPy-γ-PyPyPyPy-β-Dp, AcImImPy-γ-PyPyPy-G-Dp, H₂N-β-PyPyPy-γ-ImIm-Py-β-β-β-β-PyPyPy-γ-ImPy-β-Dp,ImPyPyPy-γ-ImPyPyPy-β-Dp, PyPyPy-γ-ImImPy-β-Dp, PyPyPy-γ-ImImPy-G-Dp,ImPyPy-β-ImImPyPy-γ-ImImPyPy-β-Dp, ImPyPy-β-PyPyPy-β-Dp,ImImPyPy-γ-ImImPyPy-β-Dp, ImPyPy-γ-β-β-β-β-Dp, ImPyPy-γ-β-PyPy-β-Dp,ImPyPy-γ-ImPyPy-β-PyPyPy-G-Ta-EDTA, H₂N-γ-ImPyPyPy-γ-PyPyPyPy(G-Dp)-COOH, ImImImIm-γ-PyPyPyPy-β-Dp,ImPyPyPy-β-ImImPyPy-γ-ImImPyPy-β-Ta-EDTA, ImPyPyPy-γ-PyPyPyPy-β-Dp,DM-γ-ImPyPy-γ-ImPyPy-β-ED, ImPyPyPy-γ-PyPyPyPy-Ta,ImPyPyPy-γ-PyPyPyPy-Ta-EDTA, ImPyPyPy-γ-ImPyPyPy-β-Ta,ImPyPyPy-γ-ImPyPyPy-β-Ta-EDTA, ImPyPyPy-γ-ImImImPy-β-Ta,ImPyPyPy-γ-ImImImPy-β-PyPyPyPy-β-Ta, ImPyPy-γ-^(m) PyPyPy-β-Dp, ImPy^(m)Py-γ-PyPyPy-β-Dp, EDTA-γ-ImPyPy-G-PyPyPy-G-Dp,EDTA-γ-ImPyPy-G-PyPyPy-G-Ta-EDTA, and EDTA-γ-ImPyPy-β-PyPyPy-G-Ta-EDTA.31. The method of anyone of claims 1 and 23, wherein said polyamide orsaid conjugate recognizes double stranded DNA by forming hydrogen bondswith bases in the minor groove of the DNA.
 32. The method of claim 31wherein wherein said polyamide or said conjugate is capable ofrecognizing double stranded DNA by interaction with the minor groove ofthe DNA.
 33. The method of claim 1 wherein said polyamide is selectedfrom the group consisting of analogs of Netropsin and Distamycin A. 34.The method of claim 1, wherein said polyamide is a straight chainpolyamide.
 35. A method of using a polyamide or a conjugate of claims20, 24, an anti-viral, anti-bacterial, or anti-tumor compound comprisingadministering a therapeutically effective amount of the compound orconjugate to an organism in need of such treatment, wherein saidpolyamide-oligonucleotide conjugate of claim 40 is used as an anti-viralcompound and not as an anti-bacterial or anti-tumour compound.
 36. Themethod of claim 1 or 23, wherein the step of adding the protective andreactive amino acids is performed at a rate of about 72 minutes peramino acid residue.
 37. The method of claim 1 or 23, further comprisingperforming in situ neutralization by adding DIEA simultaneously with thestep of sequentially adding the protected and reactive amino acids. 38.The method of claim 1 or 23, further comprising performing highperformance liquid chromatography.
 39. The method of any one of claims 1and 23, wherein said purifying is performed by high performance liquidchronatography.
 40. A method of using the polyamide of any one of claims1, and 24, to inhibit DNA binding by a DNA binding protein, comprisingproviding said polyamide to a cell containing said DNA binding protein.41. The method of claim 40, wherein said method results in regulation ofexpression of a gene in said cell.
 42. The method of claim 1, whereinsaid carboxamide groups are substituted.
 43. The method of any one ofclaims 10-12, wherein R₂ is methyl.
 44. The method of claim 1 whereinthe polyamide is cleaved from the resin by amminolysis.
 45. The polyamidof claim 20, wherein said polyamide comprises a core structure selectedfrom the group consisting of:(a) ImPyPyPyPyPyPy; (b) ImPyPyXPyPyPy;wherein X is selected from the group consisting of G, β, and Py, and Yis G or β; (c) Im Py₃ γImPy₃ ; (d) Im Py₃ γPy₄ ; and (e)ImImPyPyγPyPyPyPy.
 46. The polyamide of claim 20 or 22 wherein each ofsaid monomer units is a carboxamide pair.
 47. The method of claim 23wherein said polyamide contains imidazole and pyrrole carboxamidegroups.
 48. The method of any one of claims 1, 23, and 27, wherein theamino acids of the polyamide are deprotected.
 49. The method of claim 39wherein said ester links a polyamide to a resin.